Inhibitors of the epidermal growth factor receptor-heat shock protein 90 binding interaction

ABSTRACT

Provided herein are compounds that inhibit a binding interaction between an epidermal growth factor receptor (EGFR) and a heat shock protein 90 (HSP90), as well as compositions, e.g., pharmaceutical compositions, comprising the same, and related kits. In some embodiments, the compound is an antibody or antibody analog, and, in other embodiments, the compound is a peptide or peptide analog. Also provided are methods of using the compounds, including methods of increasing degradation of an EGFR, methods of treating cancer, and methods of sensitizing tumors to radiation therapy.

This application claims the benefit of U.S. Provisional PatentApplication No. 61/425,100 filed Dec. 20, 2010. The provisionalapplication is hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant No.CA131290, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 71 kilobytes ACII (Text) file named“45776_SeqListing.txt,” created on Dec. 7, 2010.

BACKGROUND

The Epidermal Growth Factor Receptor (EGFR) is a validated therapeutictarget for the treatment of many cancers. However, the inhibition ofEGFR has proven effective only in a limited subset of patients¹.

The limitations of these cancer therapeutics may be due to the fact thatthe therapeutics aim to inhibit the tyrosine kinase activity of EGFR. Arecent study showed that the knockdown of EGFR with small interferingRNA led to cell death in an autophagic process, independently of EGFRreceptor tyrosine kinase activity². Accordingly, the inhibition of EGFRtyrosine kinase activity alone is likely insufficient to causecytotoxicity of EGFR driven tumors.

A further drawback of some EGFR-targeted cancer therapeutics currentlyused in the clinical setting is that drug resistance often developsafter initial use of the therapeutic. For example, while non-small celllung cancer is initially sensitive to erlotinib, resistance developsupon subsequent administrations of this drug.

In view of the foregoing, there exists a need for a cancer therapeuticthat targets EGFR in a manner other than inhibition of EGFR tyrosinekinase activity. There also exists a need for a therapeutic that treatscancer without drug resistance developing after initial use.

SUMMARY

For the first time, it is shown herein that EGFR binds to Heat ShockProtein 90 (HSP90), and that specific disruption of this bindinginteraction induces EGFR degradation. Also, for the first time, it isshown herein that a particular region of EGFR is important to thebinding interaction with HSP90. Based in part on these data, compoundsthat inhibit the binding interaction between EGFR and HSP90 have beenmade and have successfully demonstrated induction of selective EGFRdegradation, cytotoxicity, and tumor regression.

Accordingly, the present disclosures provide compounds that inhibit abinding interaction between an EGFR and an HSP90. As further discussedherein, in some embodiments, the compound takes form of an antibody, orantibody analog, a peptide, or peptide analog.

Also provided by the present disclosures is a composition, e.g., apharmaceutical composition, comprising a compound that inhibits abinding interaction between an EGFR and an HSP90. Kits comprising acompound that inhibits a binding interaction between an EGFR and anHSP90, optionally, in combination with a cancer therapeutic, also areprovided herein.

Methods of using the compounds of the present disclosures are furtherprovided herein. For example, a method of inhibiting a bindinginteraction between an EGFR and an HSP90 in a cell is provided. Themethod comprises contacting the cell with a compound that inhibits thebinding interaction between an EGFR and an HSP90 in an amount effectiveto inhibit the binding interaction.

The present disclosures further provide methods of increasingdegradation of an EGFR in a cell. The method comprises contacting thecell with a compound that inhibits the binding interaction between anEGFR and an HSP90 in an amount effective to increase the degradation.

The present disclosures furthermore provide methods of treating cancerin a subject. The method comprises administering to the subject apharmaceutical composition comprising a compound that inhibits thebinding interaction between an EGFR and an HSP90 in an amount effectiveto treat the cancer.

Further provided herein are methods of sensitizing tumors to radiationtherapy, chemotherapy, or to both radiation therapy and chemotherapy, ina subject. The method comprises administering to the subject apharmaceutical composition comprising a compound that inhibits thebinding interaction between an EGFR and an HSP90 in an amount effectiveto sensitize the tumor to the therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates that EGFR is a client protein of HSP90. (a)Interaction between HSP90 and EGFR was assessed by immunoprecipitation.(b) This interaction was confirmed in UMSCC11B cells upon transfectionwith FLAG-HSP90 followed by FLAG immunoprecipitation and immunoblottingwith EGFR antibody. Treatment with GA blocked interaction between EGFRand HSP90 (c) Specificity of the interaction was confirmed in CHO cellsby transfection of full length EGFR followed by HSP90immunoprecipitation and immunoblotting for EGFR and ErbB2. (d) A directinteraction between EGFR and HSP90 was confirmed by GST pull down assay.

FIG. 2 represents immunoblots that demonstrate the interaction betweenHSP90 and EGFR. Immunoprecipitation of HSP90 and EGFR was followed byimmunoblotting with EGFR and HSP90.

FIG. 3 demonstrates the sequence-dependent interaction between EGFR andHSP90. (a) Diagram of EGFR, showing various mutants around the regioncomprising amino acids 768-773 (S768A, S768D, S768I, 768 SVDNPH 773 (SEQID NO: 15) to 768 NHVPSD 773 (SEQ ID NO: 35), D770G, DN770-771AA, andP772G). (b) All the mutant constructs shown in FIG. 2 a along with thevector or WT-EGFR were expressed in CHO cells, followed byimmunoprecipitation with HSP90 and then immunoblotting for both EGFR,HSP90 and HSP70 to detect any potential differences in the EGFR-HSP90interaction as well as EGFR expression. Results indicate that severalmutants in 768-773 region in EGFR had a large impact on EGFR stability,which was directly correlated to its interaction with HSP90. (c) Todetermine whether the cause of reduced protein interaction in the caseof 768-773 EGFR was reduced interaction between EGFR and HSP90 or simplyreduced expression of EGFR, cells were treated with a proteasomeinhibitor, MG132, for 8 hours then HSP90 was immunoprecipitated followedby immunoblotted for EGFR and HSP90. Our data indicate that MG132treatment restored 768-73 EGFR protein levels (see input); however, theinteraction between HSP90 and 768-73 EGFR remained minimal when comparedto the WT-EGFR. These results demonstrate that the decrease in 768-773EGFR-HSP90 interaction is not due simply to lowered expression of768-773 EGFR. d) We then hypothesized that 768-773 EGFR would be lessstable compared to WT EGFR due to its decreased binding with HSP90.Therefore we determined the half-life of each form of EGFR usingCycloheximide treatment (50 μg/ml) and found that 768-773 EGFR issignificantly less stable than WT-EGFR.

FIG. 4 demonstrates the penetration and stability of cell permeablepeptides in cultured cells. Biotinylated peptide fused with the HIV-TATsequence was designed from FIG. 3 a. (a) UMSCC1 cells were incubatedwith 100 μg/ml biotinylated HIV-TAT conjugated peptide for 1 h. Cellswere washed, fixed and then incubated with Streptavidin-Alexa Fluor 562reagent to visualize the internalized peptide using a fluorescencemicroscope. (b) Stability of the peptide was assessed by fixing cells atdifferent times after treatment as visualized as described above.

FIG. 5 demonstrates the molecular interaction of peptides with HSP90 andeffects on EGFR-HSP90 interaction, EGFR degradation and cell survival.(a) To determine if the lead peptide interacts directly with tumorHSP90, cell lysates from cancer or normal cells were prepared by 3freeze-thaw cycles and then incubated with 3 μg/ml of specific ornon-specific biotin coupled peptides for 2 hours. The peptide wasaffinity purified using Streptavidin-agarose beads, and bound HSP90 wasdetected by immunoblotting for HSP90. Result suggests that specificpeptide has greater affinity for tumor HSP90 compared to HSP90 fromnormal cells, and that this interaction with EGFR was minimal. (b) Theeffect of the treatment with specific and non-specific peptide onHSP90-EGFR interaction was determined by IP of HSP90 followed by IB withEGFR at 24 hours post treatment. Result show that treatment withspecific peptide reduced HSP90-EGFR interaction in UMSCC1 and H1975cells but had no effect on normal cells as assessed by densitometricanalysis and expressed relative to untreated control. Geldanamycin (GA,an HSP90 inhibitor used as a positive control), induced degradation ofEGFR in all cell lines treated (see input) and also reduced the level ofinteraction between EGFR and HSP90. The increased levels of HSP70 uponGA treatment indicate inhibition of HSP90 activity. (c) Specificity ofthe peptide treatment compared to GA was evaluated in tumor and normalcells as described in FIG. 3 b, by assessing the levels of EGFR and PARPcleavage (marker of apoptotic cell death) at 72 hours post treatment,(d) cell survival was assessed using colony formation assay.

FIG. 6 demonstrates the effect of peptide concentration on HSP90 bindingand effect on HSP90's ability to bind with ATP. (a) UMSCC1 cell lysatewas incubated with three concentrations of specific or non-specificpeptide (10, 30 and 100 μg/ml) followed by immunoblotting withanti-HSP90 antibody. Result shows that the amount of HSP90 pull-downedwas proportional to an increase in the concentration of specificpeptide. The non-specific peptide showed minimal interaction with HSP90.(b) The effect of EGFR specific peptide on HSP90's ability to bind withATP was assessed. Cisplatin treatment which is known to activate HSP90which enhances HSP90 binding with ATP was used as a positive control.Three days after treatment cell lysate was incubated with ATP-Sepharosebeads to pull down active HSP90 protein. Bound protein was eluted inSDS-PAGE buffer, and the levels were determined by immunoblot analysis.Peptide treatment had no effect on HSP90's ability to bind with ATP.

FIG. 7 demonstrates the delivery, detection and effects of cellpermeable peptides in UMSCC1 xenografts. Delivery of the peptide to theUMSCC1 xenografts was assessed after injection of peptides (8 mg/kg,i.p.), 3 days later the tumors were removed, cryosections wereimmunostained for EGFR, and the internalized biotinylated peptide wasvisualized by Streptavidin Alexa Fluor 562 reagent. The tumors from themice treated with the specific peptide, showed reduced levels of EGFR inthe areas positive for the peptide as detected by the Streptavidin AlexaFluor 562 reagent.

FIG. 8 demonstrates the efficacy and specificity of peptide treatment onHSP90 and EGFR levels in tumor and adjacent normal tissue. (a) Todetermine if EGFR level was affected in the UMSCC1 xenograft tumor, micewere injected with either DMSO, specific, or non-specific biotinylatedpeptide (8 mg/kg) intraperitoneally. Three weeks after the injection,tumors (white arrow) along with some adjacent normal tissue (yellowarrow) were harvested. Immunostaining was performed for EGFR and HSP90.HSP90 levels were not affected by either treatment either in tumor or inthe adjacent skin tissue. EGFR expression was reduced in the tumortreated with specific peptide relative to normal adjacent skin cells.(b) The effect on tumor growth was also assessed after treatment withtwo injections separated by 3 days. Tumor volume was recorded everyother day during this period, (c) Time to tumor volume doubling wasplotted for each treatment condition using Kaplan-Meier method and (d)Comparisons between any two treatment groups was analyzed by log ranktests.

FIG. 9 demonstrates the effect of peptide treatment on tumor cellproliferation and cell death. (a) 18 days post treatment tumors wereharvested and immunostained to assess the mechanism of tumor responseusing either ApopTag (apoptosis), LC3B (autophagy) and ki67 (cellproliferation). A representative field for H&E staining from each groupis also included. There was no difference in either apoptosis or cellproliferation; however, the specific peptide treated tumor showed anincrease in the punctate staining of LC3B, indicating activation ofautophagy pathway. (b) These results were confirmed in HeLa-LC3B-GFPcells, grown in culture, where chloroquine was used as a positivecontrol.

FIG. 10 demonstrates the effect of peptide treatment on micro-bloodvessel density.

FIG. 11( a-c) shows the efficacy of peptide treatment in NCI-H1975xenografts.

FIG. 12 shows the effect of peptide treatment on capillary sprouting.

DETAILED DESCRIPTION

Inhibitors of EGFR-HSP90 Binding Interactions

Provided herein are compounds that inhibit a protein-protein bindinginteraction between an epidermal growth factor receptor (EGFR) and anheat shock protein 90 (HSP90). The compounds of the present disclosuresmay be considered as inhibitors of EGFR binding to an HSP90 and/orinhibitors of HSP90 binding to an EGFR. In some embodiments, thecompounds are competitive binding inhibitors. In certain aspects, thecompounds bind to the site of EGFR to which HSP90 binds. In certainaspects, the compounds bind to the site of HSP90 to which EGFR binds. Inalternative embodiments, the compounds are non-competitive bindinginhibitors. In certain aspects, the compounds inhibit the bindinginteraction between EGFR and HSP90, yet the compounds bind to a site ofEGFR other than the site to which HSP90 binds or the compounds bind to asite of HSP90 other than the site to which EGFR binds.

The inhibition provided by the compounds of the present disclosures maynot be a 100% or complete inhibition or abrogation of the bindinginteraction between the EGFR and HSP90. Rather, there are varyingdegrees of inhibition of which one of ordinary skill in the artrecognizes as having a potential benefit or therapeutic effect. In thisrespect, the compounds of the present disclosures may inhibit thebinding interaction between an EGFR and an HSP90 to any amount or level.In exemplary embodiments, the compound provides at least or about a 10%inhibition (e.g., at least or about a 20% inhibition, at least or abouta 30% inhibition, at least or about a 40% inhibition, at least or abouta 50% inhibition, at least or about a 60% inhibition, at least or abouta 70% inhibition, at least or about a 80% inhibition, at least or abouta 90% inhibition, at least or about a 95% inhibition, at least or abouta 98% inhibition) of the binding between EGFR and HSP90. In someembodiments, the compound completely abrogates the binding interactionbetween the EGFR and the HSP90, such that no EGFR-HSP90 bindingcomplexes are detectable in a sample obtained from a subject, asmeasured by, for example, Western blotting, immunohistochemistry, andthe like.

In some embodiments of the present disclosures, the compounds inhibitthe binding interaction between a wild-type human EGFR and an HSP90. Insome aspects, the compound inhibits the binding interaction betweenHSP90 and a wild-type human EGFR comprising an amino acid sequence ofhuman EGFR isoform a (the amino acid sequence of which is accessiblefrom the National Center for Biotechnology Information (NCBI) ProteinDatabase as Accession No. NP_(—)005219). In some aspects, the compoundinhibits the binding interaction between HSP90 and a wild-type humanEGFR comprising an amino acid sequence of human EGFR isoform b (theamino acid sequence of which is accessible from the NCBI ProteinDatabase as Accession No. NP_(—)958439.1). In some aspects, the compoundinhibits the binding interaction between HSP90 and a wild-type humanEGFR comprising an amino acid sequence of human EGFR isoform c (theamino acid sequence of which is accessible from the NCBI ProteinDatabase as Accession No. NP_(—)958440.1). In some aspects, the compoundinhibits the binding interaction between HSP90 and a wild-type humanEGFR comprising an amino acid sequence of human EGFR isoform d (theamino acid sequence of which is accessible from the NCBI ProteinDatabase as Accession No. NP_(—)958441). The amino acid sequences ofeach wild-type human EGFR isoform are provided herein as SEQ ID NOs:1-4.

In other embodiments, the compounds inhibit the binding interactionbetween a mutant EGFR and an HSP90, wherein the mutant EGFR comprises anamino acid sequence which differs from any wild-type human EGFR aminoacid sequence recognized in the art (e.g., the amino acid sequences ofhuman EGFR isoforms a-d). In some aspects, the compound inhibits thebinding interaction between HSP90 and the T790M EGFR mutant comprisingthe amino acid sequence of SEQ ID NO: 5 with the Thr at position 790mutated to a Met. In other aspects, the compound inhibits the bindinginteraction between HSP90 and the L858R EGFR mutant comprising the aminoacid sequence of SEQ ID NO: 34. In alternative aspects, the compoundinhibits the binding interaction between HSP90 and the EGFRvIII mutantin which amino acids 6 to 273 are deleted and thus comprises the aminoacid sequence of SEQ ID NO: 6.

In some embodiments, the compounds inhibit the binding interactionbetween a wild-type human HSP90 and an EGFR. In some aspects, thecompound inhibits the binding interaction between an EGFR and an HSP90comprising an amino acid sequence of SEQ ID NO: 7.

In exemplary aspects, the compound is an antibody, an antibody analog, apeptide, a peptide analog (e.g., peptoid, peptidomimetic), a nucleicacid molecule encoding any of the antibodies or peptides, or analogsthereof, or a small molecule compound (e.g., small molecule compoundrationally designed based on any of the antibodies or peptides describedherein).

Antibodies and Analogs Thereof.

In some embodiments of the present disclosures, the compound thatinhibits a binding interaction between an EGFR and HSP90 comprises anantibody, or antigen binding fragment thereof. In some embodiments ofthe present disclosures, the compound that inhibits a bindinginteraction between an EGFR and HSP90 is provided as an antibody, orantigen binding fragment thereof. The antibody may be any type ofimmunoglobulin known in the art. In exemplary embodiments, the antibodyis an antibody of isotype IgA, IgD, IgE, IgG, or IgM. Also, the antibodyin some embodiments is a monoclonal antibody. In other embodiments, theantibody is a polyclonal antibody.

In some embodiments, the antibody is a naturally-occurring antibody,e.g., an antibody isolated and/or purified from a mammal, e.g., mouse,rabbit, goat, horse, chicken, hamster, human, and the like. In thisregard, the antibody may be considered as a mammalian antibody, e.g., amouse antibody, rabbit antibody, goat antibody, horse antibody, chickenantibody, hamster antibody, human antibody, and the like. Methods ofproducing naturally-occurring antibodies are known in the art, some ofwhich are described further herein under the section entitled “Methodsof Antibody Production.”

In some embodiments, the antibody is a genetically-engineered antibody,e.g., a single chain antibody, a humanized antibody, a chimericantibody, a CDR-grafted antibody, an antibody which includes portions ofCDR sequences specific for EGFR or HSP90, a humaneered antibody, abispecific antibody, a trispecific antibody, and the like. Geneticengineering techniques also provide the ability to make fully humanantibodies in a non-human source.

In some aspects, the genetically-engineered antibody is a single chainantibody (SCA) specific for EGFR or HSP90. In particular aspects, theSCA binds to the site of EGFR to which HSP90 binds or the SCA binds tothe site of HSP90 to which EGFR binds. In exemplary aspects, the SCAbinds to an epitope as further described herein under the sectionentitled “Epitopes.” Methods of making SCAs are known in the art. See,for example, Davis et al., Nature Biotechnology 9: 165-169 (1991).

In some aspects, the antibody is a chimeric antibody. The term “chimericantibody” is used herein to refer to an antibody containing constantdomains from one species and the variable domains from a second, or moregenerally, containing stretches of amino acid sequence from at least twospecies. In particular aspects, the chimeric antibody binds to the siteof EGFR to which HSP90 binds or the chimeric antibody binds to the siteof HSP90 to which EGFR binds. In exemplary aspects, the chimericantibody binds to an epitope as further described herein under thesection entitled “Epitopes.”

In some aspects, the antibody is a humanized antibody. The term“humanized” when used in relation to antibodies refers to antibodieshaving at least CDR regions from a non-human source which are engineeredto have a structure and immunological function more similar to truehuman antibodies than the original source antibodies. For example,humanizing can involve grafting CDR from a non-human antibody, such as amouse antibody, into a human antibody. Humanizing also can involveselect amino acid substitutions to make a non-human sequence look morelike a human sequence. In particular aspects, the humanized antibodybinds to the site of EGFR to which HSP90 binds or the humanized antibodybinds to the site of HSP90 to which EGFR binds. In exemplary aspects,the humanized antibody binds to an epitope as further described hereinunder the section entitled “Epitopes.”

Use of the terms “chimeric or humanized” herein is not meant to bemutually exclusive, and rather, is meant to encompass chimericantibodies, humanized antibodies, and chimeric antibodies that have beenfurther humanized. Except where context otherwise indicates, statementsabout (properties of, uses of, testing of, and so on) chimericantibodies of the present disclosures apply to humanized antibodies ofthe present disclosures, and statements about humanized antibodies ofthe present disclosures pertain also to chimeric antibodies. Likewise,except where context dictates, such statements also should be understoodto be applicable to antibodies and antigen binding fragments of suchantibodies of the present disclosures.

In some aspects, the antibody is a CDR-grafted antibody specific forEGFR or HSP90. In particular aspects, the CDR-grafted antibody binds tothe site of EGFR to which HSP90 binds or the CDR-grafted antibody bindsto the site of HSP90 to which EGFR binds. In exemplary aspects, theCDR-grafted antibody binds to an epitope as further described hereinunder the section entitled “Epitopes.” Methods of making CDR-graftedantibodies are known in the art. See, for example, Lo, Benny, AntibodyEngineering: Methods and Protocols, Volume 248 (2004), which isincorporated by reference in its entirety.

In some aspects, the antibody is a bispecific or trispecific antibodyspecific for EGFR or HSP90. In particular aspects, the bispecific ortrispecific antibody binds to the site of EGFR to which HSP90 binds orthe bispecific or trispecific antibody binds to the site of HSP90 towhich EGFR binds. In exemplary aspects, the bispecific or trispecificantibody binds to an epitope as further described herein under thesection entitled “Epitopes.” Methods of making bispecific or trispecificantibodies are known in the art. See, for example, Marvin and Zhu, ActaPharmacologica Sinica 26: 649-658 (2005) and U.S. Pat. No. 6,551,592.

In some aspects, the antibody is a Humaneered™ antibody. Humaneeringtechnology is a proprietary method of KaloBios Pharmaceuticals, Inc.(San Francisco, Calif.) for converting non human antibodies intoengineered human antibodies. Humaneered™ antibodies are high affinity,and highly similar to human germline antibody sequences.

In some embodiments, the antibody has a level of affinity or avidity forthe EGFR which is sufficient to prevent HSP90 from binding to the EGFR.In some embodiments, the antibody has a level of affinity or avidity forthe HSP90 which is sufficient to prevent EGFR from binding HSP90.Therefore in some embodiments, the affinity constant, K_(a), (which isthe inverterd dissocation constant, K_(d)) of the antibody of thepresent disclosures for the EGFR is greater than the K_(a) of HSP90 forthe EGFR. Alternatively, in some embodiments, the K_(a) of the antibodyof the present disclosures for the HSP90 is greater than that of EGFRfor HSP90. Binding constants, including dissociation constants, may bedetermined by methods known in the art, including, for example, methodswhich utilize the principles of surface plasmon resonance, e.g., methodsutilizing a Biacore™ system.

In some embodiments, the antibody is in monomeric form, while in otherembodiments, the antibody is conjugated to one or more antibodies (e.g.,each of which recognize the same epitope of the first antibody).Accordingly, in some aspects, the antibody is in polymeric, oligomeric,or multimeric form. In certain embodiments in which the antibodycomprises two or more distinct antigen binding regions fragments, theantibody is considered bispecific, trispecific, or multi-specific, orbivalent, trivalent, or multivalent, depending on the number of distinctepitopes that are recognized and bound by the antibody.

Antigen Binding Fragments

In some aspects of the present disclosures, the compound which inhibitsa binding interaction between an EGFR and HSP90 is an antigen bindingfragment of an antibody. The antigen binding fragment (also referred toherein as “antigen binding portion”) may be an antigen binding fragmentof any of the antibodies described herein. The antigen binding fragmentcan be any part of an antibody that has at least one antigen bindingsite, including, but not limited to, Fab, F(ab′)₂, dsFv, sFv, diabodies,triabodies, bis-scFvs, fragments expressed by a Fab expression library,domain antibodies, VhH domains, V-NAR domains, VH domains, VL domains,and the like. Antibody fragments of the invention, however, are notlimited to these exemplary types of antibody fragments.

A domain antibody comprises a functional binding unit of an antibody,and can correspond to the variable regions of either the heavy (V_(H))or light (V_(L)) chains of antibodies. A domain antibody can have amolecular weight of approximately 13 kDa, or approximately one-tenth ofa full antibody. Domain antibodies may be derived from full antibodiessuch as those described herein. The antigen binding fragments in someembodiments are monomeric or polymeric, bispecific or trispecific,bivalent or trivalent.

Antibody fragments that contain the antigen binding, or idiotype, of theantibody molecule may be generated by techniques known in the art. Forexample, such fragments include, but are not limited to, the F(ab′)₂fragment which may be produced by pepsin digestion of the antibodymolecule; the Fab′ fragments which may be generated by reducing thedisulfide bridges of the F(ab′)₂ fragment, and the two Fab′ fragmentswhich may be generated by treating the antibody molecule with papain anda reducing agent.

A single-chain variable region fragment (sFv) antibody fragment, whichconsists of a truncated Fab fragment comprising the variable (V) domainof an antibody heavy chain linked to a V domain of a light antibodychain via a synthetic peptide, can be generated using routinerecombinant DNA technology techniques (see, e.g., Janeway et al.,supra). Similarly, disulfide-stabilized variable region fragments (dsFv)can be prepared by recombinant DNA technology (see, e.g., Reiter et al.,Protein Engineering, 7, 697-704 (1994)).

Recombinant antibody fragments, e.g., scFvs, can also be engineered toassemble into stable multimeric oligomers of high binding avidity andspecificity to different target antigens. Such diabodies (dimers),triabodies (trimers) or tetrabodies (tetramers) are well known in theart, see e.g., Kortt et al., Biomol Eng. 2001 18:95-108, (2001) andTodorovska et al., J Immunol Methods. 248:47-66, (2001).

Bispecific antibodies (bscAb) are molecules comprising two single-chainFv fragments joined via a glycine-serine linker using recombinantmethods. The V light-chain (V_(L)) and V heavy-chain (V_(H)) domains oftwo antibodies of interest in exemplary embodiments are isolated usingstandard PCR methods. The V_(L) and V_(H) cDNA's obtained from eachhybridoma are then joined to form a single-chain fragment in a two-stepfusion PCR. Bispecific fusion proteins are prepared in a similar manner.Bispecific single-chain antibodies and bispecific fusion proteins areantibody substances included within the scope of the present invention.Exemplary bispecific antibodies are taught in U.S. Patent ApplicationPublication No. 2005-0282233A1 and International Patent ApplicationPublication No. WO 2005/087812, both applications of which areincorporated herein by reference in their entirety.

Methods of Antibody or Antigen Binding Fragment Production

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Harlow andLane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA.Janeway et al. (eds.), Immunobiology, 5^(th) Ed., Garland Publishing,New York, N.Y. (2001)).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide of the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. In some aspects, ananimal used for production of anti-antisera is a non-human animalincluding rabbits, mice, rats, hamsters, goat, sheep, pigs or horses.Because of the relatively large blood volume of rabbits, a rabbit is apreferred choice for production of polyclonal antibodies. In anexemplary method for generating a polyclonal antisera immunoreactivewith the chosen EGFR epitope, 50 μg of EGFR antigen is emulsified inFreund's Complete Adjuvant for immunization of rabbits. At intervals of,for example, 21 days, 50 μg of epitope are emulsified in Freund'sIncomplete Adjuvant for boosts. Polyclonal antisera may be obtained,after allowing time for antibody generation, simply by bleeding theanimal and preparing serum samples from the whole blood.

Monoclonal antibodies for use in the invention may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include but are not limited tothe hybridoma technique originally described by Koehler and Milstein(Nature 256: 495-497, 1975), the human B-cell hybridoma technique(Kosbor et al., Immunol Today 4:72, 1983; Cote et al., Proc Natl AcadSci 80: 2026-2030, 1983) and the EBV-hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New YorkN.Y., pp 77-96, (1985).

Briefly, in exemplary embodiments, to generate monoclonal antibodies, amouse is injected periodically with recombinant EGFR against which theantibody is to be raised (e.g., 10-20 μg emulsified in Freund's CompleteAdjuvant). The mouse is given a final pre-fusion boost of an EGFRpolypeptide in PBS, and four days later the mouse is sacrificed and itsspleen removed. The spleen is placed in 10 ml serum-free RPMI 1640, anda single cell suspension is formed by grinding the spleen between thefrosted ends of two glass microscope slides submerged in serum-free RPMI1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100units/ml penicillin, and 100 μg/ml streptomycin (RPMI) (Gibco, Canada).The cell suspension is filtered through sterile 70-mesh Nitex cellstrainer (Becton Dickinson, Parsippany, N.J.), and is washed twice bycentrifuging at 200 g for 5 minutes and resuspending the pellet in 20 mlserum-free RPMI. Splenocytes taken from three naive Balb/c mice areprepared in a similar manner and used as a control. NS-1 myeloma cells,kept in log phase in RPMI with 11% fetal bovine serum (FBS) (HycloneLaboratories, Inc., Logan, Utah) for three days prior to fusion, arecentrifuged at 200 g for 5 minutes, and the pellet is washed twice.

Spleen cells (1×10⁸) are combined with 2.0×10⁷ NS-1 cells andcentrifuged, and the supernatant is aspirated. The cell pellet isdislodged by tapping the tube, and 1 ml of 37° C. PEG 1500 (50% in 75 mMHepes, pH 8.0) (Boehringer Mannheim) is added with stirring over thecourse of 1 minute, followed by the addition of 7 ml of serum-free RPMIover 7 minutes. An additional 8 ml RPMI is added and the cells arecentrifuged at 200 g for 10 minutes. After discarding the supernatant,the pellet is resuspended in 200 ml RPMI containing 15% FBS, 100 μMsodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco),25 units/ml IL-6 (Boehringer Mannheim) and 1.5×10⁶ splenocytes/ml andplated into 10 Corning flat-bottom 96-well tissue culture plates(Corning, Corning N.Y.).

On days 2, 4, and 6, after the fusion, 100 μl of medium is removed fromthe wells of the fusion plates and replaced with fresh medium. On day 8,the fusion is screened by ELISA, testing for the presence of mouse IgGbinding to EGFR as follows. Immulon 4 plates (Dynatech, Cambridge,Mass.) are coated for 2 hours at 37° C. with 100 ng/well of EGFR dilutedin 25 mM Tris, pH 7.5. The coating solution is aspirated and 200 μl/wellof blocking solution (0.5% fish skin gelatin (Sigma) diluted in CMF-PBS)is added and incubated for 30 min. at 37° C. Plates are washed threetimes with PBS with 0.05% Tween 20 (PBST) and 50 μl culture supernatantis added. After incubation at 37° C. for 30 minutes, and washing asabove, 50 μl of horseradish peroxidase conjugated goat anti-mouseIgG(fc) (Jackson ImmunoResearch, West Grove, Pa.) diluted 1:3500 in PBSTis added. Plates are incubated as above, washed four times with PBST,and 100 μl substrate, consisting of 1 mg/ml o-phenylene diamine (Sigma)and 0.1 μl/ml 30% H₂O₂ in 100 mM Citrate, pH 4.5, are added. The colorreaction is stopped after 5 minutes with the addition of 50 μl of 15%H₂SO₄. A₄₉₀ is read on a plate reader (Dynatech).

Selected fusion wells are cloned twice by dilution into 96-well platesand visual scoring of the number of colonies/well after 5 days. Themonoclonal antibodies produced by hybridomas are isotyped using theIsostrip system (Boehringer Mannheim, Indianapolis, Ind.).

When the hybridoma technique is employed, myeloma cell lines may beused. Such cell lines suited for use in hybridoma-producing fusionprocedures preferably are non-antibody-producing, have high fusionefficiency, and enzyme deficiencies that render them incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas). For example, where the immunizedanimal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1,Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/15XX0 Bul; forrats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266,GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection withcell fusions. It should be noted that the hybridomas and cell linesproduced by such techniques for producing the monoclonal antibodies arecontemplated to be novel compositions of the present disclosures.

Depending on the host species, various adjuvants may be used to increaseimmunological response. Such adjuvants include but are not limited toFreund's, mineral gels such as aluminum hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG(bacilli Calmette-Guerin) and Corynebacterium parvum are potentiallyuseful human adjuvants.

Alternatively, other methods, such as EBV-hybridoma methods (Haskard andArcher, J. Immunol. Methods, 74(2), 361-67 (1984), and Roder et al.,Methods Enzymol., 121, 140-67 (1986)), and bacteriophage vectorexpression systems (see, e.g., Huse et al., Science, 246, 1275-81(1989)) are known in the art. Further, methods of producing antibodiesin non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806,5,569,825, and 5,714,352, and U.S. Patent Application Publication No.2002/0197266 A1).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al (Proc Natl Acad Sci 86: 3833-3837; 1989), and Winter G andMilstein C (Nature 349: 293-299, 1991).

Phage display furthermore can be used to generate the antibody of thepresent disclosures. In this regard, phage libraries encodingantigen-binding variable (V) domains of antibodies can be generatedusing standard molecular biology and recombinant DNA techniques (see,e.g., Sambrook et al. (eds.), Molecular Cloning, A Laboratory Manual,3^(rd) Edition, Cold Spring Harbor Laboratory Press, New York (2001)),Phage encoding a variable region with the desired specificity areselected for specific binding to the desired antigen, and a complete orpartial antibody is reconstituted comprising the selected variabledomain. Nucleic acid sequences encoding the reconstituted antibody areintroduced into a suitable cell line, such as a myeloma cell used forhybridoma production, such that antibodies having the characteristics ofmonoclonal antibodies are secreted by the cell (see, e.g., Janeway etal., supra, Huse et al., supra, and U.S. Pat. No. 6,265,150). Relatedmethods also are described in U.S. Pat. No. 5,403,484; U.S. Pat. No.5,571,698; U.S. Pat. No. 5,837,500; U.S. Pat. No. 5,702,892. Thetechniques described in U.S. Pat. No. 5,780,279; U.S. Pat. No.5,821,047; U.S. Pat. No. 5,824,520; U.S. Pat. No. 5,855,885; U.S. Pat.No. 5,858,657; U.S. Pat. No. 5,871,907; U.S. Pat. No. 5,969,108; U.S.Pat. No. 6,057,098; U.S. Pat. No. 6,225,447,

Antibodies can be produced by transgenic mice that are transgenic forspecific heavy and light chain immunoglobulin genes. Such methods areknown in the art and described in, for example U.S. Pat. Nos. 5,545,806and 5,569,825, and Janeway et al., supra.

Methods for generating humanized antibodies are well known in the artand are described in detail in, for example, Janeway et al., supra, U.S.Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No.0239400 B1, and United Kingdom Patent No. 2188638. Humanized antibodiescan also be generated using the antibody resurfacing technologydescribed in U.S. Pat. No. 5,639,641 and Pedersen et al., J. Mol. Biol,235, 959-973 (1994).

Techniques developed for the production of “chimeric antibodies”, thesplicing of mouse antibody genes to human antibody genes to obtain amolecule with appropriate antigen specificity and biological activity,can be used (Morrison et al., Proc Natl Acad Sci 81: 6851-6855, 1984;Neuberger et al., Nature 312: 604-608, 1984; Takeda et al., Nature 314:452-454; 1985). Alternatively, techniques described for the productionof single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce EGFR- or HSP90-specific single chain antibodies.

A preferred chimeric or humanized antibody has a human constant region,while the variable region, or at least a CDR, of the antibody is derivedfrom a non-human species. Methods for humanizing non-human antibodiesare well known in the art. (see U.S. Pat. Nos. 5,585,089, and5,693,762). Generally, a humanized antibody has one or more amino acidresidues introduced into its framework region from a source which isnon-human. Humanization can be performed, for example, using methodsdescribed in Jones et al. (Nature 321: 522-525, 1986), Riechmann et al.,(Nature, 332: 323-327, 1988) and Verhoeyen et al. (Science239:1534-1536, 1988), by substituting at least a portion of a rodentcomplementarity-determining region (CDRs) for the corresponding regionsof a human antibody. Numerous techniques for preparing engineeredantibodies are described, e.g., in Owens and Young, J. Immunol. Meth.,168:149-165 (1994). Further changes can then be introduced into theantibody framework to modulate affinity or immunogenicity.

Likewise, using techniques known in the art to isolate CDRs,compositions comprising CDRs are generated. Complementarity determiningregions are characterized by six polypeptide loops, three loops for eachof the heavy or light chain variable regions. The amino acid position ina CDR is defined by Kabat et al., “Sequences of Proteins ofImmunological Interest,” U.S. Department of Health and Human Services,(1983), which is incorporated herein by reference. For example,hypervariable regions of human antibodies are roughly defined to befound at residues 28 to 35, from 49-59 and from residues 92-103 of theheavy and light chain variable regions (Janeway and Travers,Immunobiology, 2^(nd) Edition, Garland Publishing, New York, (1996)).The murine CDR also are found at approximately these amino acidresidues. It is understood in the art that CDR regions may be foundwithin several amino acids of these approximated residues set forthabove. An immunoglobulin variable region also consists of four“framework” regions surrounding the CDRs (FR1-4). The sequences of theframework regions of different light or heavy chains are highlyconserved within a species, and are also conserved between human andmurine sequences.

Compositions comprising one, two, and/or three CDRs of a heavy chainvariable region or a light chain variable region of a monoclonalantibody are generated. Techniques for cloning and expressing nucleotideand polypeptide sequences are well-established in the art (see e.g.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Edition,Cold Spring Harbor, N.Y. (1989)). The amplified CDR sequences areligated into an appropriate plasmid. The plasmid comprising one, two,three, four, five and/or six cloned CDRs optionally contains additionalpolypeptide encoding regions linked to the CDR.

Framework regions (FR) of a murine antibody are humanized bysubstituting compatible human framework regions chosen from a largedatabase of human antibody variable sequences, including over twelvehundred human VH sequences and over one thousand VL sequences. Thedatabase of antibody sequences used for comparison is downloaded fromAndrew C. R. Martin's KabatMan web page (rubic.rdg.ac.uk/abs/). TheKabat method for identifying CDR provides a means for delineating theapproximate CDR and framework regions from any human antibody andcomparing the sequence of a murine antibody for similarity to determinethe CDRs and FRs. Best matched human VH and VL sequences are chosen onthe basis of high overall framework matching, similar CDR length, andminimal mismatching of canonical and VH/VL contact residues. Humanframework regions most similar to the murine sequence are insertedbetween the murine CDR. Alternatively, the murine framework region maybe modified by making amino acid substitutions of all or part of thenative framework region that more closely resemble a framework region ofa human antibody.

Additionally, another useful technique for generating antibodies for usein the present invention may be one which uses a rational design typeapproach. The goal of rational design is to produce structural analogsof biologically active polypeptides or compounds with which theyinteract (agonists, antagonists, inhibitors, peptidomimetics, bindingpartners, etc.). In one approach, one would generate a three-dimensionalstructure for the antibodies or an epitope binding fragment thereof.This could be accomplished by x-ray crystallography, computer modelingor by a combination of both approaches. An alternative approach,“alanine scan,” involves the random replacement of residues throughoutmolecule with alanine, and the resulting affect on function determined.

It also is possible to solve the crystal structure of the specificantibodies. In principle, this approach yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies to afunctional, pharmacologically active antibody. As a mirror image of amirror image, the binding site of anti-idiotype would be expected to bean analog of the original antigen. The anti-idiotype could then be usedto identify and isolate additional antibodies from banks of chemically-or biologically-produced peptides.

Chemically constructed bispecific antibodies may be prepared bychemically cross-linking heterologous Fab or F(ab′)₂ fragments by meansof chemicals such as heterobifunctional reagentsuccinimidyl-3-(2-pyridyldithiol)-propionate (SPDP, Pierce Chemicals,Rockford, Ill.). The Fab and F(ab′)₂ fragments can be obtained fromintact antibody by digesting it with papain or pepsin, respectively(Karpovsky et al., J. Exp. Med. 160:1686-701, 1984; Titus et al., J.Immunol., 138:4018-22, 1987).

Methods of testing antibodies for the ability to bind to the epitope ofthe EGFR regardless of how the antibodies are produced are known in theart and include any antibody-antigen binding assay, such as, forexample, radioimmunoassay (RIA), ELISA, Western blot,immunoprecipitation, and competitive inhibition assays (see, e.g.,Janeway et al., infra, and U.S. Patent Application Publication No.2002/0197266 A1).

Aptamers

In some embodiments, the compound that inhibits a binding interactionbetween EGFR and HSP90 is an analog of an antibody. In some aspects, thecompound is an aptamer. Recent advances in the field of combinatorialsciences have identified short polymer sequences (e.g., oligonucleicacid or peptide molecules) with high affinity and specificity to a giventarget. For example, SELEX technology has been used to identify DNA andRNA aptamers with binding properties that rival mammalian antibodies,the field of immunology has generated and isolated antibodies orantibody fragments which bind to a myriad of compounds and phage displayhas been utilized to discover new peptide sequences with very favorablebinding properties. Based on the success of these molecular evolutiontechniques, it is certain that molecules can be created which bind toany target molecule. A loop structure is often involved with providingthe desired binding attributes as in the case of: aptamers which oftenutilize hairpin loops created from short regions without complimentarybase pairing, naturally derived antibodies that utilize combinatorialarrangement of looped hyper-variable regions and new phage displaylibraries utilizing cyclic peptides that have shown improved resultswhen compared to linear peptide phage display results. Thus, sufficientevidence has been generated to suggest that high affinity ligands can becreated and identified by combinatorial molecular evolution techniques.For the present disclosures, molecular evolution techniques can be usedto isolate compounds specific for the EGFRs or HSP90s described hereinthat inhibit the binding interaction between EGFR and HSP90. For more onaptamers, see, generally, Gold, L., Singer, B., He, Y. Y., Brody. E.,“Aptamers As Therapeutic And Diagnostic Agents,” J. Biotechnol. 74:5-13(2000). Relevant techniques for generating aptamers may be found in U.S.Pat. No. 6,699,843, which is incorporated by reference in its entirety.

Epitopes

By “epitope” as used herein is meant the region of or within the EGFR orHSP90 which is bound by the compound, e.g., the antibody, the antigenbinding fragment, the aptamer. In some embodiments, the epitope is alinear epitope. By “linear epitope” as used herein refers to the regionof or within the EGFR or HSP90 which is bound by the compound, whichregion is composed of contiguous amino acids of the amino acid sequenceof the EGFR or HSP90. The amino acids of a linear epitope are located inclose proximity to each other in the primary structure of the antigenand the secondary and/or tertiary structure(s) of the antigen. Forexample, when the antigen, e.g., EGFR or HSP90, is in its properlyfolded state (e.g., its native conformation), the contiguous amino acidsof the linear epitope are located in close proximity to one another.

In other aspects, the epitope of the binding construct is aconformational epitope. By “conformational epitope” is meant an epitopewhich is composed of amino acids which are located in close proximity toone another only when the EGFR or HSP90 is in its properly folded state,but are not contiguous amino acids of the amino acid sequence of theEGFR or HSP90.

In some embodiments of the present disclosures, the compound thatinhibits a binding interaction between an EGFR and HSP90 binds to anepitope of an EGFR. In some aspects, the epitope to which the compoundbinds is within the kinase domain of EGFR (e.g., amino acids 688 to 955of SEQ ID NO: 1). In some aspects, the compound that inhibits a bindinginteraction between an EGFR and HSP90 binds to an epitope within aminoacids 761-781 of the EGFR sequence (SEQ ID NO: 1). In further aspects,the compound that inhibits a binding interaction between an EGFR andHSP90 binds to an epitope within amino acids 768-773 of the SEQ ID NO:1, amino acids 768-775 of SEQ ID NO: 1, or amino acids 776-781 of SEQ IDNO: 1.

In some embodiments, the compound that inhibits a binding interactionbetween an EGFR and HSP90 binds to an epitope comprising the amino acidsequence DNPH (SEQ ID NO: 13) or RLLGIC (SEQ ID NO: 14). In specificaspects, the compound binds to an epitope comprising the amino acidsequence SVDNPH (SEQ ID NO: 15), SVDNPHV (SEQ ID NO: 16), or SVDNPHVX(SEQ ID NO: 17), wherein X is Cys, Ser, Ala, Gly, Val.

In yet other embodiments, the compound that inhibits the bindinginteraction between an EGFR and HSP90 binds to an epitope comprising theamino acid sequence of any of the peptides or peptide analogs describedherein, which peptides or peptide analogs are described herein as acompound that inhibits the binding interaction between an EGFR andHSP90.

Peptides

In some embodiments of the present disclosures, the compound thatinhibits a binding interaction between an EGFR and HSP90 is a peptidecomprising at least four amino acids connected via peptide bonds. Insome aspects, the peptide is about 4 to about 50 amino acids in length.In some aspects, the compound is about 6 to about 25 amino acids inlength. In some aspects, the compound is about 8 to about 12 amino acidsin length. In some embodiments, the peptide is an 8-mer.

Fragments of EGFR

In some embodiments, the peptide that inhibits a binding interactionbetween an EGFR and HSP90 is a fragment of a human wild-type EGFR, e.g.,any of those disclosed herein. In some aspects, the compound is afragment of wild-type human EGFR isoform a which is provided herein asSEQ ID NO: 1. In specific aspects, the compound comprises 4 to 10consecutive amino acids of amino acids 688 to 955 of SEQ ID NO: 1, whichportion of SEQ ID NO: 1 represents the kinase domain of the EGFR. Infurther aspects, the compound comprises 4 to 10 consecutive amino acidsfrom amino acids 761-781 of the SEQ ID NO: 1. In yet further aspects,the compound comprises the amino acid sequence DNPH (SEQ ID NO: 13) orRLLGIC (SEQ ID NO: 14). In some embodiments, the compound comprises theamino acid sequence SVDNPH (SEQ ID NO: 15), SVDNPHV (SEQ ID NO: 16), orSVDNPHVC (SEQ ID NO: 18).

Derivatives

In some embodiments, the peptide that inhibits a binding interactionbetween an EGFR and HSP90 comprises an amino acid sequence which isbased on the amino acid sequence of a human wild-type EGFR, or afragment thereof, but differs at one or more (e.g., two, three, four,five, six, seven, eight, nine, ten, or more) amino acid positions, whenaligned with the human wild-type EGFR sequence, or fragment thereof.

In some embodiments, the peptide that inhibits a binding interactionbetween an EGFR and HSP90 comprises an amino acid sequence which has atleast 25% sequence identity to the amino acid sequence of a humanwild-type EGFR, e.g., SEQ ID NO: 1, or a fragment thereof (e.g., afragment of about 4 to about 10 contiguous amino acids of SEQ ID NO: 8or 9). In some embodiments, the compound comprises an amino acidsequence which is at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or has greater than 95% sequence identity to SEQ IDNO: 1, or a fragment thereof (e.g., a fragment of about 4 to about 10contiguous amino acids of SEQ ID NO: 8 or 9).

In exemplary embodiments, the compound comprises an amino acid sequencewhich is based on the EGFR fragment SVDNPH (SEQ ID NO: 15). In someaspects, the compound comprises SVDNPHVX (SEQ ID NO: 17), wherein X isany amino acid. In particular aspects, X is the native residue whichfollows this sequence in the wild-type EGFR amino acid sequence (namely,Cys). In other aspects, X is a non-native residue or is an amino acidother than Cys. In particular aspects, X is an amino acid which issimilar in size to Cys, but lacks the sulfur atom. In exemplary aspects,X is Ser, Gly, Val, or Ala.

In other exemplary embodiments, the compound comprises an amino acidsequence of SVDNPH (SEQ ID NO: 15) with up to two amino acidsubstitutions. In some aspects, the amino acid substitution(s) occur(s)at one or two of the positions 1, 2, 3, 4, 5, and 6. In particularaspects, the amino acid substitution(s) occur(s) at one or two of thepositions 1, 3, and 5 of SVDNPH (SEQ ID NO: 15). In some aspects, thecompound comprises the amino acid sequence XVXNXH (SEQ ID NO: 19),XVXNPH (SEQ ID NO: 20), XVDNPH (SEQ ID NO: 21), SVXNXH (SEQ ID NO: 22),SVXNPH (SEQ ID NO: 23), or SVDNXH (SEQ ID NO: 24), wherein each Xrepresents any amino acid. In some aspects, the X is the native residueof the wild-type EGFR amino acid sequence. In other aspects, the X is anon-native residue of the wild-type EGFR amino acid sequence. Inexemplary aspects, the compound comprises S, D, I, or A at position 1 ofSEQ ID NO: 19. In some aspects, the compound comprises D, A, or G atposition 3 of SEQ ID NO: 19. In some aspects, the compound comprises Por G at position 5 of SEQ ID NO: 19. In some aspects, the compoundcomprises an amino acid sequence selected from the group consisting of:AVDNPH (SEQ ID NO: 25), DVDNPH (SEQ ID NO: 26), IVDNPH (SEQ ID NO: 27),SVGNPH (SEQ ID NO: 28), SVDNGH (SEQ ID NO: 29), and SVAAPH (SEQ ID NO:30).

Cyclized or Bridged Compounds

In some embodiments, the peptide that inhibits a binding interactionbetween an EGFR and HSP90 is a cyclized peptide or a peptide thatcomprises an intramolecular bridge which links the side chains of twoamino acids of the compound. In some embodiments, the intramolecularbridge is a bridge which connects two parts of the peptide vianoncovalent bonds, including, for example, van der Waals interactions,hydrogen bonds, ionic bonds, hydrophobic interactions, dipole-dipoleinteractions, and the like. In this regard, the peptide in certainaspects comprises a non-covalent intramolecular bridge. In someembodiments, the intramolecular bridge is a bridge which connects twoparts of the peptide via covalent bonds. In this regard, the peptide incertain aspects comprises a covalent intramolecular bridge.

Non-Covalent Intramolecular Bridges

In some embodiments, the non-covalent intramolecular bridge is a saltbridge. In exemplary embodiments, the peptide is bridged between twoamino acids: one of the amino acids of the peptide is an amino acid ofFormula I and the other amino acid of the peptide.

In some embodiments, the non-covalent intramolecular bridge is ahydrophobic bridge. In accordance with one embodiment, the compound isstabilized through the incorporation of hydrophobic amino acids atpositions j and j+5 or i and i+4. For instance, i can be Tyr and i+4 canbe either Val or Leu; i can be Phe and i+4 can be Met; or i can be Pheand i+4 can be Ile. It should be understood that, for purposes herein,the above amino acid pairings can be reversed, such that the indicatedamino acid at position i could alternatively be located at i+4, whilethe i+4 amino acid can be located at the i position. It should also beunderstood that suitable amino acid pairings can be formed for j andj+5.

Covalent Intramolecular Bridge

In some embodiments, the covalent intramolecular bridge is a lactam ringor lactam bridge. The size of the lactam ring can vary depending on thelength of the amino acid side chains, and in one embodiment the lactamis formed by linking the side chains of an ornithine to a aspartic acidside chain. Lactam bridges and methods of making the same are known inthe art. See, for example, Houston, Jr., et al., J Peptide Sci 1:274-282 (2004). In some embodiments, the compound comprises a modifiedsequence of a fragment of SEQ ID NO: 1 and a lactam bridge between i andi+4, wherein i is as defined herein above.

In some embodiments, the covalent intramolecular bridge is a lactone.Suitable methods of making a lactone bridge are known in the art. See,for example, Sheehan et al., J Am Chem Soc 95: 875-879 (1973).

In some aspects, olefin metathesis is used to cross-link the compoundusing an all-hydrocarbon cross-linking system. The compound in thisinstance comprises α-methylated amino acids bearing olefinic side chainsof varying length and configured with either R or S stereochemistry atthe j and j+5 or i and i+4 positions. In some embodiments, the olefinicside comprises (CH₂)n, wherein n is any integer between 1 to 6. In someembodiments, n is 3 for a cross-link length of 8 atoms. In someembodiments, n is 2 for a cross-link length of 6 atoms. Suitable methodsof forming such intramolecular bridges are described in the art. See,for example, Schafmeister et al., J. Am. Chem. Soc. 122: 5891-5892(2000) and Walensky et al., Science 305: 1466-1470 (2004). Inalternative embodiments, the compound comprises O-allyl Ser residues,which are bridged together via ruthenium-catalyzed ring closingmetathesis. Such procedures of cross-linking are described in, forexample, Blackwell et al., Angew, Chem., Int. Ed. 37: 3281-3284 (1998).

In specific aspects, use of the unnatural thio-dialanine amino acid,lanthionine, which has been widely adopted as a peptidomimetic ofcystine, is used to cross-link one turn of the alpha helix. Suitablemethods of lanthionine-based cyclization are known in the art. See, forinstance, Matteucci et al., Tetrahedron Letters 45: 1399-1401 (2004);Mayer et al., J. Peptide Res. 51: 432-436 (1998); Polinsky et al., J.Med. Chem. 35: 4185-4194 (1992); Osapay et al., J. Med. Chem. 40:2241-2251 (1997); Fukase et al., Bull. Chem. Soc. Jpn. 65: 2227-2240(1992); Harpp et al., J. Org. Chem. 36: 73-80 (1971); Goodman and Shao,Pure Appl. Chem. 68: 1303-1308 (1996); and Osapay and Goodman, J. Chem.Soc. Chem. Commun. 1599-1600 (1993).

In some embodiments, α, ω-diaminoalkane tethers, e.g.,1,4-diaminopropane and 1,5-diaminopentane) between two Glu residues atpositions i and i+7 are used to stabilize the compound. Such tetherslead to the formation of a bridge 9-atoms or more in length, dependingon the length of the diaminoalkane tether. Suitable methods of producingpeptides cross-linked with such tethers are described in the art. See,for example, Phelan et al., J. Am. Chem. Soc. 119: 455-460 (1997).

In yet other embodiments, a disulfide bridge is used to cross-linkcompound. Alternatively, a modified disulfide bridge in which one orboth sulfur atoms are replaced by a methylene group resulting in anisosteric macrocyclization is used to stabilize the alpha helix of thecompound. Suitable methods of modifying peptides with disulfide bridgesor sulfur-based cyclization are described in, for example, Jackson etal., J. Am. Chem. Soc. 113: 9391-9392 (1991) and Rudinger and Jost,Experientia 20: 570-571 (1964).

In yet other embodiments, the compound is stabilized via the binding ofmetal atom by two His residues or a His and Cys pair positioned at j andj+3, or i and i+4. The metal atom can be, for example, Ru(III), Cu(II),Zn(II), or Cd(II). Such methods of metal binding-based alpha helixstabilization are known in the art. See, for example, Andrews and Tabor,Tetrahedron 55: 11711-11743 (1999); Ghadiri et al., J. Am. Chem. Soc.112: 1630-1632 (1990); and Ghadiri et al., J. Am. Chem. Soc. 119:9063-9064 (1997).

The compound may alternatively be stabilized through other means ofpeptide cyclizing, which means are reviewed in Davies, J. Peptide. Sci.9: 471-501 (2003). The compound may be stabilized via the formation ofan amide bridge, thioether bridge, thioester bridge, urea bridge,carbamate bridge, sulfonamide bridge, and the like. For example, athioester bridge can be formed between the C-terminus and the side chainof a Cys residue. Alternatively, a thioester can be formed via sidechains of amino acids having a thiol (Cys) and a carboxylic acid (e.g.,Asp, Glu). In another method, a cross-linking agent, such as adicarboxylic acid, e.g., suberic acid (octanedioic acid), etc. canintroduce a link between two functional groups of an amino acid sidechain, such as a free amino, hydroxyl, thiol group, and combinationsthereof.

Spacing/Size of Bridge

In some embodiments, the intramolecular bridge (e.g., non-covalentintramolecular bridge, covalent intramolecular bridge) is formed betweentwo amino acids that are 3 amino acids apart, e.g., amino acids atpositions i and i+4. In specific embodiments, wherein the amino acids atpositions i and i+4 are joined by an intramolecular bridge, the size ofthe linker is about 8 atoms, or about 7-9 atoms.

In other embodiments, the intramolecular bridge is formed between twoamino acids that are four amino acids apart, e.g., amino acids atpositions j and j+5. In specific embodiments, wherein amino acids atpositions j and j+5 are joined by an intramolecular bridge, the size ofthe linker is about 6 atoms, or about 5 to 7 atoms.

In yet other embodiments, the intramolecular bridge is formed betweentwo amino acids that are five amino acids apart, e.g., amino acids atpositions k and k+6.

Amino Acids Involved in Intramolecular Bridges

Examples of amino acid pairings that are capable of bonding (covalentlyor non-covalently) to form a six-atom linking bridge include Orn andAsp, Glu and an amino acid of Formula I, wherein n is 2, andhomoglutamic acid and an amino acid of Formula I, wherein n is 1,wherein Formula I is:

Examples of amino acid pairings that are capable of bonding to form aseven-atom linking bridge include Orn-Glu (lactam ring); Lys-Asp(lactam); or Homoser-Homoglu (lactone). Examples of amino acid pairingsthat may form an eight-atom linker include Lys-Glu (lactam); Homolys-Asp(lactam); Orn-Homoglu (lactam); 4-aminoPhe-Asp (lactam); or Tyr-Asp(lactone). Examples of amino acid pairings that may form a nine-atomlinker include Homolys-Glu (lactam); Lys-Homoglu (lactam);4-aminoPhe-Glu (lactam); or Tyr-Glu (lactone). Any of the side chains onthese amino acids may additionally be substituted with additionalchemical groups. One of ordinary skill in the art can envisionalternative pairings or alternative amino acid analogs, includingchemically modified derivatives, that would create a stabilizingstructure of similar size and desired effect. For example, ahomocysteine-homocysteine disulfide bridge is 6 atoms in length and maybe further modified to provide the desired effect.

Even without covalent linkage, the amino acid pairings described above(or similar pairings that one of ordinary skill in the art can envision)may also provide added stability to the compound through non-covalentbonds, for example, through formation of salt bridges orhydrogen-bonding interactions. Accordingly, salt bridges may be formedbetween: Orn and Glu; Lys and Asp; Homo-serine and Homo-glutamate; Lysand Glu; Asp and Arg; Homo-Lys and Asp; Orn and Homo-Glutamate;4-aminoPhe and Asp; Tyr and Asp; Homo-Lys and Glu; Lys and Homo-Glu;4-aminoPhe and Glu; or Tyr and Glu. In some embodiments, the compoundcomprises a salt bridge between any of the following pairs of aminoacids: Orn and Glu; Lys and Asp; Lys and Glu; Asp and Arg; Homo-Lys andAsp; Orn and Homo-Glutamate; Homo-Lys and Glu; and Lys and Homo-Glu.Salt bridges may be formed between other pairs of oppositely chargedside chains. See, e.g., Kallenbach et al., Role of the Peptide Bond inProtein Structure and Folding, in The Amide Linkage: StructuralSignificance in Chemistry, Biochemistry, and Materials Science, JohnWiley & Sons, Inc. (2000).

Exemplary Cyclized or Bridged Compounds

In some aspects, the compound that inhibits a binding interactionbetween an EGFR and HSP90 comprises an intramolecular bridge which linkstwo amino acids separated by 3, 4, or 5 amino acids in the amino acidsequence of the compound. In some aspects, the intramolecular bridge isa disulfide bridge, a dithioether bridge, a carba analog bridge, or alactam bridge. In specific aspects, when the intramolecular bridge is adithioether, the dithioether comprises the structure —S(CH₂)_(n)S—,wherein n is 1, 2, 3, 4, or 5. In specific aspects, when theintramolecular bridge is carba analog bridge, the carba analog bridgecomprises a C3 to C10 alkyl chain.

In some aspects, when the intramolecular bridge is a lactam bridge, thelactam is formed between the side chains of an amino acid of Formula Iand an amino acid of Formula II, wherein Formula I is:

and Formula II is:

In specific embodiments, the amino acid of Formula I is Lys or Orn. Inspecific embodiments, the amino acid of Formula II is Asp or Glu.

In some embodiments, the compound comprises the amino acid sequenceXDNPHX (SEQ ID NO: 32), wherein the side chains of the amino acids atpositions 1 and 6 are covalently linked by the intramolecular bridge. Insome aspects, each of the amino acids at positions 1 and 6 is Cys. Inspecific aspects, the side chains of the amino acids at positions 1 and6 are linked by a disulfide bond or a dithioether. In alternativeaspects, the intramolecular bridge is a lactam and one of the aminoacids at positions 1 and 6 is an amino acid of Formula I and the otheramino acid is an amino acid of Formula II. In some aspects, the compoundcomprises the amino acid sequence SXDNPHXX (SEQ ID NO: 33), wherein theX at position 8 is C, S, or A.

Additional Peptide Modifications

In alternative or additional embodiments of the present disclosures, thepeptide is glycosylated, amidated, carboxylated, phosphorylated,esterified, N-acylated, acetylated, or converted into an acid additionsalt and/or optionally dimerized or polymerized, or conjugated, asfurther described herein.

In some aspects, the first amino acid of the peptide is acetylated atthe N-terminus in which the N-terminal alpha —NH₂ group of an unmodifiedpeptide is converted into —COCH₃. In alternative or additional aspects,the last amino acid of the peptide is amidated at the C-terminus inwhich the C-terminal —COOH group of an unmodified peptide is convertedinto an —NH₂.

Peptide Analogs

In some embodiments, the compound is a peptide analog having a structurebased on one of the peptides disclosed herein (the “parent peptide”) butdiffers from the parent peptide in one or more respects. Accordingly, asappreciated by one of ordinary skill in the art the teachings of theparent peptides provided herein may also be applicable the peptideanalogs.

In some aspects, the peptide analog comprises the structure of a parentpeptide, except that the peptide analog comprises one or morenon-peptide bonds in place of peptide bond(s). In exemplary aspects, thepeptide analog comprises in place of a peptide bond, an ester bond, anether bond, a thioether bond, an amide bond, and the like. In someaspects, the peptide analog is a depsipeptide comprising an esterlinkage in place of a peptide bond.

In some aspects, the peptide analog comprises the structure of a parentpeptide described herein, except that the peptide analog comprises oneor more amino acid substitutions, e.g., one or more conservative aminoacid substitutions. Conservative amino acid substitutions are known inthe art, and include amino acid substitutions in which one amino acidhaving certain physical and/or chemical properties is exchanged foranother amino acid that has the same chemical or physical properties.For instance, the conservative ammo acid substitution may be an acidicamino acid substituted for another acidic amino acid (e.g., Asp or Glu),an amino acid with a nonpolar side chain substituted for another aminoacid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met,Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for anotherbasic amino acid (Lys, Arg, etc.), an amino acid with a polar side chainsubstituted for another amino acid with a polar side chain (Asn, Cys,Gln, Ser, Thr, Tyr, etc.), etc.

In some aspects, the peptide analog comprises one or more syntheticamino acids, e.g., an amino acid non-native to a mammal. Synthetic aminoacids include β-alanine (β-Ala), N-α-methyl-alanine (Me-Ala),aminobutyric acid (Abu), γ-aminobutyric acid (γ-Abu), aminohexanoic acid(ε-Ahx), aminoisobutyric acid (Aib), aminomethylpyrrole carboxylic acid,aminopiperidinecarboxylic acid, aminoserine (Ams),aminotetrahydropyran-4-carboxylic acid, arginine N-methoxy-N-methylamide, β-aspartic acid (β-Asp), azetidine carboxylic acid,3-(2-benzothiazolyl)alanine, α-tert-butylglycine,2-amino-5-ureido-n-valeric acid (citrulline, Cit), β-Cyclohexylalanine(Cha), acetamidomethyl-cysteine, diaminobutanoic acid (Dab),diaminopropionic acid (Dpr), dihydroxyphenylalanine (DOPA),dimethylthiazolidine (DMTA), γ-Glutamic acid (γ-Glu), homoserine (Hse),hydroxyproline (Hyp), isoleucine N-methoxy-N-methyl amide,methyl-isoleucine (MeIle), isonipecotic acid (Isn), methyl-leucine(MeLeu), methyl-lysine, dimethyl-lysine, trimethyl-lysine,methanoproline, methionine-sulfoxide (Met(O)), methionine-sulfone(Met(O₂)), norleucine (Nle), methyl-norleucine (Me-Nle), norvaline(Nva), ornithine (Orn), para-aminobenzoic acid (PABA), penicillamine(Pen), methylphenylalanine (MePhe), 4-Chlorophenylalanine (Phe(4-Cl)),4-fluorophenylalanine (Phe(4-F)), 4-nitrophenylalanine (Phe(4-NO₂)),4-cyanophenylalanine ((Phe(4-CN)), phenylglycine (Phg),piperidinylalanine, piperidinylglycine, 3,4-dehydroproline,pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec),O-Benzyl-phosphoserine, 4-amino-3-hydroxy-6-methylheptanoic acid (Sta),4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA),4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA),1,2,3,4,-tetrahydro-isoquinoline-3-carboxylic acid (Tic),tetrahydropyranglycine, thienylalanine (Thi), O-benzyl-phosphotyrosine,O-Phosphotyrosine, methoxytyrosine, ethoxytyrosine,O-(bis-dimethylamino-phosphono)-tyrosine, tyrosine sulfatetetrabutylamine, methyl-valine (MeVal), and alkylated3-mercaptopropionic acid.

In some embodiments, the peptide analog comprises one or morenon-conservative amino acid substitutions and the peptide analog stillfunctions to a similar extent, the same extent, or an improved extent asthe parent peptide. In certain aspects, the peptide analog comprisingone or more non-conservative amino acid substitutions inhibits thebinding interaction between EGFR and HSP90 to an extent better than theparent peptide.

In some embodiments, and/or one or more amino acid insertions ordeletions, in reference to the parent peptide described herein. In someembodiments, the peptide analog comprises an insertion of one or moreamino acids at the N- or C-terminus in reference to the parent peptide.In some embodiments, the peptide analog comprises a deletion of one ormore amino acids at the N- or C-terminus in reference to the parentpeptide. In these aspects, the peptide analog still functions to asimilar extent, the same extent, or an improved extent as the parentpeptide to inhibit the binding interaction between EGFR and HSP90.

In some aspects, the peptide analog is a peptidomimetic. Peptidomimeticsas well as methods of making the same are known in the art. See, forexample, Advances in Amino Acid Mimetics and Peptidomimetics, Volumes 1and 2, ed., Abell, A., JAI Press Inc., Greenwich, Conn., 2006. In someaspects, the peptidomimetic is a D-peptide peptidomimetic comprisingD-isomer amino acids. In some aspects, the peptidomimetic is a peptoidin which the side chain of an amino acid is connected to the alphanitrogen atom of the peptide backbone. Methods of making peptoids areknown in the art. See, e.g., Zuckermann et al., JACS 114(26):10646-10647 (1992) and Design, Synthesis, and Evaluation of NovelPeptoids, Fowler, Sarah, University of Wisconsin-Madison, 2008. In someaspects, the peptidomimetic is a β-peptide comprising β amino acidswhich have their amino group bonded to the β-cargon rather than thealpha carbon. Methods of making β-peptides are known in the art. See,for example, Seebach et al., Helvetica Chimica Acta 79(4): 913-941(1996).

Pharmaceutically Acceptable Salts

With regard to the present disclosures, the compounds that inhibit abinding interaction between an EGFR and HSP90, (collectively referred tohereinafter as “active agents”) in some aspects is in the form of asalt, e.g., a pharmaceutically acceptable salt. Such salts can beprepared in situ during the final isolation and purification of theactive agent or separately prepared by reacting a free base functionwith a suitable acid. Examples of acids which can be employed to formpharmaceutically acceptable acid addition salts include, for example, aninorganic acid, e.g., hydrochloric acid, hydrobromic acid, sulphuricacid, and phosphoric acid, and an organic acid, e.g., oxalic acid,maleic acid, succinic acid, and citric acid.

Representative acid addition salts include, but are not limited toacetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphor sulfonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate (isothionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalene sulfonate, oxalate, palmitoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate, and undecanoate.

Basic addition salts also can be prepared in situ during the finalisolation and purification of the active agent, or by reacting acarboxylic acid-containing moiety with a suitable base such as thehydroxide, carbonate, or bicarbonate of a pharmaceutically acceptablemetal cation or with ammonia or an organic primary, secondary, ortertiary amine. Pharmaceutically acceptable salts include, but are notlimited to, cations based on alkali metals or alkaline earth metals suchas lithium, sodium, potassium, calcium, magnesium, and aluminum salts,and the like, and nontoxic quaternary ammonia and amine cationsincluding ammonium, tetramethylammonium, tetraethylammonium,methylammonium, dimethylammonium, trimethylammonium, triethylammonium,diethylammonium, and ethylammonium, amongst others. Other representativeorganic amines useful for the formation of base addition salts include,for example, ethylenediamine, ethanolamine, diethanolamine, piperidine,piperazine, and the like.

Further, basic nitrogen-containing groups can be quaternized with suchactive agents as lower alkyl halides such as methyl, ethyl, propyl, andbutyl chlorides, bromides, and iodides; long chain halides such asdecyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides;arylalkyl halides like benzyl and phenethyl bromides and others. Wateror oil-soluble or dispersible products are thereby obtained.

Isolated and Purified

The compounds of the present disclosures that inhibit a bindinginteraction between an EGFR and an HSP90 can be isolated and/orpurified. The term “isolated” as used herein means having been removedfrom its natural environment. The term “purified” as used herein meanshaving been increased in purity, wherein “purity” is a relative term,and not to be necessarily construed as absolute purity. In exemplaryaspects, the purity of the compound (e.g., in the composition) is atleast or about 50%, at least or about 60%, at least or about 70%, atleast or about 80%, at least or about 90%, at least or about 95%, or atleast or about 98% or is about 100%.

Methods of Making Peptides

The peptides of the present disclosure may be obtained by methods knownin the art. Suitable methods of de novo synthesizing peptides aredescribed in, for example, Chan et al., Fmoc Solid Phase PeptideSynthesis, Oxford University Press, Oxford, United Kingdom, 2005;Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc.,2000; Epitope Mapping, ed. Westwood et al., Oxford University Press,Oxford, United Kingdom, 2000; and U.S. Pat. No. 5,449,752. Additionalexemplary methods of making the peptides of the present disclosures areset forth herein.

In some embodiments, the peptides described herein are commerciallysynthesized by companies, such as Synpep (Dublin, Calif.), PeptideTechnologies Corp. (Gaithersburg, Md.), Multiple Peptide Systems (SanDiego, Calif.), Peptide 2.0 Inc. (Chantilly, Va.), and American PeptideCo. (Sunnyvale, Calif.). In this respect, the peptides can be synthetic,recombinant, isolated, and/or purified.

Also, in some aspects, the peptides are recombinantly produced using anucleic acid encoding the amino acid sequence of the peptide usingstandard recombinant methods. See, for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, NY, 1994.

In some embodiments, the glucagon analogs of the disclosure areisolated. The term “isolated” as used herein means having been removedfrom its natural environment. In exemplary embodiments, the analog ismade through recombinant methods and the analog is isolated from thehost cell.

In some embodiments, the glucagon analogs of the disclosure arepurified. The term “purified,” as used herein relates to the isolationof a molecule or compound in a form that is substantially free ofcontaminants which in some aspects are normally associated with themolecule or compound in a native or natural environment and means havingbeen increased in purity as a result of being separated from othercomponents of the original composition. The purified peptide or compoundinclude, for example, peptides substantially free of nucleic acidmolecules, lipids, and carbohydrates, or other starting materials orintermediates which are used or formed during chemical synthesis of thepeptides. It is recognized that “purity” is a relative term, and not tobe necessarily construed as absolute purity or absolute enrichment orabsolute selection. In some aspects, the purity is at least or about50%, is at least or about 60%, at least or about 70%, at least or about80%, or at least or about 90% (e.g., at least or about 91%, at least orabout 92%, at least or about 93%, at least or about 94%, at least orabout 95%, at least or about 96%, at least or about 97%, at least orabout 98%, at least or about 99% or is approximately 100%.

Nucleic Acids

In some embodiments of the present disclosures, the compound thatinhibits a binding interaction between an EGFR and HSP90 comprises anucleic acid comprising a nucleotide sequence encoding any of theantibodies or peptides described herein (including analogs thereof). Thenucleic acid can comprise any nucleotide sequence which encodes any ofthe antibodies, peptides, or analogs thereof. By “nucleic acid” as usedherein includes “polynucleotide,” “oligonucleotide,” and “nucleic acidmolecule,” and generally means a polymer of DNA or RNA, which can besingle-stranded or double-stranded, synthesized or obtained (e.g.,isolated and/or purified) from natural sources, which can containnatural, non-natural or altered nucleotides, and which can contain anatural, non-natural or altered internucleotide linkage, such as aphosphoroamidate linkage or a phosphorothioate linkage, instead of thephosphodiester found between the nucleotides of an unmodifiedoligonucleotide. In some embodiments, the nucleic acid does not compriseany insertions, deletions, inversions, and/or substitutions. In otherembodiments, the nucleic acid comprises one or more insertions,deletions, inversions, and/or substitutions.

In some aspects, the nucleic acids of the present disclosures arerecombinant. As used herein, the term “recombinant” refers to (i)molecules that are constructed outside living cells by joining naturalor synthetic nucleic acid segments to nucleic acid molecules that canreplicate in a living cell, or (ii) molecules that result from thereplication of those described in (i) above. For purposes herein, thereplication can be in vitro replication or in vivo replication.

The nucleic acids in some aspects are constructed based on chemicalsynthesis and/or enzymatic ligation reactions using procedures known inthe art. See, for example, Sambrook et al., supra; and Ausubel et al.,supra. For example, a nucleic acid can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed upon hybridization(e.g., phosphorothioate derivatives and acridine substitutednucleotides). Examples of modified nucleotides that can be used togenerate the nucleic acids include, but are not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridme,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substitutedadenine, 7-methylguanine, 5-methylammomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouratil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N²-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

Recombinant Expression Vector

The nucleic acids of the present disclosures in some aspects areincorporated into a recombinant expression vector. In this regard, thepresent disclosures provides recombinant expression vectors comprisingany of the presently disclosed nucleic acids. For purposes herein, theterm “recombinant expression vector” means a genetically-modifiedoligonucleotide or polynucleotide construct that permits the expressionof an mRNA, protein, polypeptide, or peptide by a host cell, when theconstruct comprises a nucleotide sequence encoding the mRNA, protein,polypeptide, or peptide, and the vector is contacted with the cell underconditions sufficient to have the mRNA, protein, polypeptide, or peptideexpressed within the cell. The vectors of the present disclosures arenot naturally-occurring as a whole. However, parts of the vectors can benaturally-occurring. The presently disclosed recombinant expressionvectors may comprise any type of nucleotides, including, but not limitedto DNA and RNA, which may be single-stranded or double-stranded,synthesized or obtained in part from natural sources, and which cancontain natural, non-natural or altered nucleotides. The recombinantexpression vectors may comprise naturally-occurring ornon-naturally-occurring internucleotide linkages, or both types oflinkages. In some aspects, the altered nucleotides or non-naturallyoccurring internucleotide linkages do not hinder the transcription orreplication of the vector.

The recombinant expression vector of the present disclosures can be anysuitable recombinant expression vector, and can be used to transform ortransfect any suitable host. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasmidsand viruses. The vector can be selected from the group consisting of thepUC series (Fermentas Life Sciences), the pBluescript series(Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.),the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λGTIO,λGTl 1, λZapII (Stratagene), λEMBL4, and λNMl 149, also can be used.Examples of plant expression vectors include pBIOl, pBI101.2, pBI101.3,pBI121 and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-Cl, pMAM and pMAMneo (Clontech). In some aspects, therecombinant expression vector is a viral vector, e.g., a retroviralvector.

The recombinant expression vectors of the present disclosures can beprepared using standard recombinant DNA techniques described in, forexample, Sambrook et al., supra, and Ausubel et al., supra. Constructsof expression vectors, which are circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from CoIEl, 2μplasmid, λ, SV40, bovine papilloma virus, and the like.

In some aspects, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

The recombinant expression vector may include one or more marker genes,which allow for selection of transformed or transfected hosts. Markergenes include biocide resistance, e.g., resistance to antibiotics, heavymetals, etc., complementation in an auxotrophic host to provideprototrophy, and the like. Suitable marker genes for the presentlydisclosed expression vectors include, for instance, neomycin/G418resistance genes, hygromycin resistance genes, histidinol resistancegenes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or normativepromoter operably linked to the nucleotide sequence encoding thepolypeptide (including functional portions and functional variantsthereof), or to the nucleotide sequence which is complementary to orwhich hybridizes to the nucleotide sequence encoding the polypeptide.The selection of promoters, e.g., strong, weak, inducible,tissue-specific and developmental-specific, is within the ordinary skillof the artisan. Similarly, the combining of a nucleotide sequence with apromoter is also within the skill of the artisan. The promoter can be anon-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV)promoter, an SV40 promoter, an RSV promoter, and a promoter found in thelong-terminal repeat of the murine stem cell virus.

The presently disclosed recombinant expression vectors may be designedfor either transient expression, for stable expression, or for both.Also, the recombinant expression vectors may be made for constitutiveexpression or for inducible expression. Further, the recombinantexpression vectors may be made to include a suicide gene.

As used herein, the term “suicide gene” refers to a gene that causes thecell expressing the suicide gene to die. The suicide gene in someaspects is a gene that confers sensitivity to an agent, e.g., a drug,upon the cell in which the gene is expressed, and causes the cell to diewhen the cell is contacted with or exposed to the agent. Suicide genesare known in the art (see, for example, Suicide Gene Therapy: Methodsand Reviews. Springer, Caroline J. (Cancer Research UK Centre for CancerTherapeutics at the Institute of Cancer Research, Sutton, Surrey, UK),Humana Press, 2004) and include, for example, the Herpes Simplex Virus(HSV) thymidine kinase (TK) gene, cytosine daminase, purine nucleosidephosphorylase, and nitroreductase.

Host Cells

The present disclosures further provides a host cell comprising any ofthe recombinant expression vectors described herein. As used herein, theterm “host cell” refers to any type of cell that can contain thepresently disclosed recombinant expression vector. The host cell in someaspects is a eukaryotic cell, e.g., plant, animal, fungi, or algae, orcan be a prokaryotic cell, e.g., bacteria or protozoa. The host cell insome aspects is a cultured cell or a primary cell, i.e., isolateddirectly from an organism, e.g., a human. The host cell in some aspectsis an adherent cell or a suspended cell, i.e., a cell that grows insuspension. Suitable host cells are known in the art and include, forinstance, DH5α E. coli cells, Chinese hamster ovarian cells, monkey VEROcells, COS cells, HEK293 cells, and the like. For purposes of amplifyingor replicating the recombinant expression vector, the host cell is insome aspects is a prokaryotic cell, e.g., a DH5α cell. For purposes ofproducing a recombinant polypeptide the host cell is in some aspects amammalian cell, e.g., a human cell. The host cell may be of any celltype, can originate from any type of tissue, and can be of anydevelopmental stage.

Also provided by the present disclosures is a population of cellscomprising at least one host cell described herein. The population ofcells in some aspects is a heterogeneous population comprising the hostcell comprising any of the recombinant expression vectors described, inaddition to at least one other cell, which does not comprise any of therecombinant expression vectors. Alternatively, in some aspects, thepopulation of cells is a substantially homogeneous population, in whichthe population comprises mainly of host cells (e.g., consistingessentially of) comprising the recombinant expression vector. Thepopulation in some aspects is a clonal population of cells, in which allcells of the population are clones of a single host cell comprising arecombinant expression vector, such that all cells of the populationcomprise the recombinant expression vector. In one embodiment of thepresent disclosures, the population of cells is a clonal populationcomprising host cells comprising a recombinant expression vector asdescribed herein.

Conjugates

In some embodiments, the compounds of the present disclosures areattached or linked or conjugated to a second moiety (e.g., aheterologous moiety, a conjugate moiety). As used herein, the term“heterologous moiety” is synonymous with “conjugate moiety” and refersto any molecule (chemical or biochemical, naturally-occurring ornon-coded) which is different from the compounds of the presentdisclosures. Exemplary heterologous moieties include, but are notlimited to, a polymer, a carbohydrate, a lipid, a nucleic acid, anoligonucleotide, a DNA or RNA, an amino acid, peptide, polypeptide,protein, therapeutic agent, (e.g., a cytotoxic agent, cytokine), or adiagnostic agent.

In some embodiments, the compounds are chemically modified with varioussubstituents. In some embodiments, the chemical modifications impartadditional desirable characteristics as discussed herein. Chemicalmodifications in some aspects take a number of different forms such asheterologous peptides, polysaccarides, lipids, radioisotopes,non-standard amino acid resides and nucleic acids, metal chelates, andvarious cytotoxic agents.

In some embodiments, the compounds are fused to heterologous peptides toconfer various properties, e.g., increased solubility and/or stabilityand/or half-life, resistance to proteolytic cleavage, modulation ofclearance, targeting to particular cell or tissue types. In someembodiments, the compound is linked to a Fc domain of IgG or otherimmunoglobulin. In some embodiments, the compound is fused to alkalinephosphatase (AP). Methods for making Fc or AP fusion constructs arefound in WO 02/060950. By fusing the compound with protein domains thathave specific properties (e.g. half life, bioavailability) it ispossible to confer these properties to the compound of the presentdisclosures.

When the compounds are peptides, they can be modified, for instance, byglycosylation, amidation, carboxylation, or phosphorylation, or by thecreation of acid addition salts, amides, esters, in particularC-terminal esters, and N-acyl derivatives, as discussed above. Thepeptides also can be modified to create peptide derivatives by formingcovalent or noncovalent complexes with other moieties. Covalently boundcomplexes can be prepared by linking the chemical moieties to functionalgroups on the side chains of amino acids comprising the peptides, or atthe N- or C-terminus.

Peptides can be conjugated to a reporter group, including, but notlimited to a radiolabel, a fluorescent label, an enzyme (e.g., thatcatalyzes a calorimetric or fluorometric reaction), a substrate, a solidmatrix, or a carrier (e.g., biotin or avidin). Examples of analogs aredescribed in WO 98/28621 and in Olofsson, et al, Proc. Nat'l. Acad. Sci.USA, 95:11709-11714 (1998), U.S. Pat. Nos. 5,512,545, and 5,474,982;U.S. Patent Application Nos. 20020164687 and 20020164710.

Cysteinyl residues most commonly are reacted with haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carbocyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-.beta.(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol,orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic orcarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylissurea; 2,4 pentanedione; and transaminase catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneare used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively. Tyrosyl residues are iodinated using ¹²⁵I or ¹³¹I toprepare labeled proteins for use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R1) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3 (4azonia 4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl andglutamyl residues are converted to asparaginyl and glutaminyl residuesby reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking thebinding construct to water-insoluble support matrixes. Such derivationmay also provide the linker that may connect adjacent binding elementsin a binding construct, or a binding elements to a heterologous peptide,e.g., a Fc fragment. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homo-bifunctional imidoesters, including disuccinimidyl esterssuch as 3,3′-dithiiobis(succinimidylpropioonate), and bifunctionalmaleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents suchas methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming cross links in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440, incorporated herein by reference, are employed forprotein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MoleculeProperties, W. H. Freeman & Co., San Francisco, pp. 79-86, 1983),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups. Such derivatives are chemicallymodified polypeptide compositions in which the binding constructpolypeptide is linked to a polymer.

In general, chemical derivatization may be performed under any suitablecondition used to react a protein with an activated polymer molecule.Methods for preparing chemical derivatives of polypeptides willgenerally comprise the steps of (a) reacting the polypeptide with theactivated polymer molecule (such as a reactive ester or aldehydederivative of the polymer molecule) under conditions whereby the bindingconstruct becomes attached to one or more polymer molecules, and (b)obtaining the reaction product(s). The optimal reaction conditions willbe determined based on known parameters and the desired result. Forexample, the larger the ratio of polymer molecules:protein, the greaterthe amount of attached polymer molecule. In some embodiments, thecompound may have a single polymer molecule moiety at the aminoterminus. (See, e.g., U.S. Pat. No. 5,234,784).

Derivatized binding constructs disclosed herein may have additionalactivities, enhanced or reduced biological activity, or othercharacteristics, such as increased or decreased half-life, as comparedto the non-derivatized molecules.

In some embodiments, the compound is directly joined to a conjugatemoiety in the absence of a linker. In alternative aspects, the compoundis indirectly connected to the conjugate moiety via one or more linkers.Whether directly joined together or indirectly joined together through alinker, the compound may be connected through covalent bonds (e.g., apeptide, ester, amide, or sulfhydryl bond) or non-covalent bonds (e.g.,via hydrophobic interaction, hydrogen bond, van der Waals bond,electrostatic or ionic interaction), or a combination thereof. Thecompound of the present disclosures and conjugate moiety may beconnected via any means known in the art, including, but not limited to,via a linker of any of the present disclosures. See, for example, thesection herein entitled “Linkers.”

Conjugates: Fc Fusions

For substituents such as an Fc region of human IgG, the fusion can befused directly to a compound of the present disclosures or fused throughan intervening sequence. For example, a human IgG hinge, CH2 and CH3region may be fused at either the N-terminus or C-terminus of a bindingconstruct to attach the Fc region. The resulting Fc-fusion constructenables purification via a Protein A affinity column (Pierce, Rockford,Ill.). Peptide and proteins fused to an Fc region can exhibit asubstantially greater half-life in vivo than the unfused counterpart. Afusion to an Fc region allows for dimerization/multimerization of thefusion polypeptide. The Fc region may be a naturally occurring Fcregion, or may be modified for superior characteristics, e.g.,therapeutic qualities, circulation time, reduced aggregation. As notedabove, in some embodiments, the compounds are conjugated, e.g., fused toan immunoglobulin or portion thereof (e.g., variable region, CDR, or Fcregion). Known types of immunoglobulins (Ig) include IgG, IgA, IgE, IgDor IgM. The Fc region is a C-terminal region of an Ig heavy chain, whichis responsible for binding to Fc receptors that carry out activitiessuch as recycling (which results in prolonged half-life), antibodydependent cell-mediated cytotoxicity (ADCC), and complement dependentcytotoxicity (CDC).

For example, according to some definitions the human IgG heavy chain Fcregion stretches from Cys226 to the C-terminus of the heavy chain. The“hinge region” generally extends from Glu216 to Pro230 of human IgG1(hinge regions of other IgG isotypes may be aligned with the IgG1sequence by aligning the cysteines involved in cysteine bonding). The Fcregion of an IgG includes two constant domains, CH2 and CH3. The CH2domain of a human IgG Fc region usually extends from amino acids 231 toamino acid 341. The CH3 domain of a human IgG Fc region usually extendsfrom amino acids 342 to 447. References made to amino acid numbering ofimmunoglobulins or immunoglobulin fragments, or regions, are all basedon Kabat et al. 1991, Sequences of Proteins of Immunological Interest,U.S. Department of Public Health, Bethesda, Md. In a relatedembodiments, the Fc region may comprise one or more native or modifiedconstant regions from an immunoglobulin heavy chain, other than CH1, forexample, the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4regions of IgE.

Suitable conjugate moieties include portions of immunoglobulin sequencethat include the FcRn binding site. FcRn, a salvage receptor, isresponsible for recycling immunoglobulins and returning them tocirculation in blood. The region of the Fc portion of IgG that binds tothe FcRn receptor has been described based on X-ray crystallography(Burmeister et al. 1994, Nature 372:379). The major contact area of theFc with the FcRn is near the junction of the CH2 and CH3 domains.Fc-FcRn contacts are all within a single Ig heavy chain. The majorcontact sites include amino acid residues 248, 250-257, 272, 285, 288,290-291, 308-311, and 314 of the CH2 domain and amino acid residues385-387, 428, and 433-436 of the CH3 domain.

Some conjugate moieties may or may not include FcγR binding site(s).FcγR are responsible for ADCC and CDC. Examples of positions within theFc region that make a direct contact with FcγR are amino acids 234-239(lower hinge region), amino acids 265-269 (B/C loop), amino acids297-299 (C′/E loop), and amino acids 327-332 (F/G) loop (Sondermann etal., Nature 406: 267-273, 2000). The lower hinge region of IgE has alsobeen implicated in the FcRI binding (Henry, et al., Biochemistry 36,15568-15578, 1997). Residues involved in IgA receptor binding aredescribed in Lewis et al., (J. Immunol. 175:6694-701, 2005). Amino acidresidues involved in IgE receptor binding are described in Sayers et al.(J Biol. Chem. 279(34):35320-5, 2004).

Amino acid modifications may be made to the Fc region of animmunoglobulin. Such variant Fc regions comprise at least one amino acidmodification in the CH3 domain of the Fc region (residues 342-447)and/or at least one amino acid modification in the CH2 domain of the Fcregion (residues 231-341). Mutations believed to impart an increasedaffinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al.2001, J. Biol. Chem. 276:6591). Other mutations may reduce binding ofthe Fc region to FcγRI, FcγRIIA, FcγRIIB, and/or FcγRIIIA withoutsignificantly reducing affinity for FcRn. For example, substitution ofthe Asn at position 297 of the Fc region with Ala or another amino acidremoves a highly conserved N-glycosylation site and may result inreduced immunogenicity with concomitant prolonged half-life of the Fcregion, as well as reduced binding to FcγR5 (Routledge et al. 1995,Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632;Shields et al. 1995, J. Biol. Chem. 276:6591). Amino acid modificationsat positions 233-236 of IgG1 have been made that reduce binding to FcγR5(Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al.1999, Eur. J. Immunol. 29:2613). Some exemplary amino acid substitutionsare described in U.S. Pat. Nos. 7,355,008 and 7,381,408, eachincorporated by reference herein in its entirety.

Heterologous Moieties: Polymers, Carbohydrates, and Lipids

In some embodiments, the heterologous moiety is a polymer. The polymermay be branched or unbranched. The polymer may be of any molecularweight. The polymer in some embodiments has an average molecular weightof between about 2 kDa to about 100 kDa (the term “about” indicatingthat in preparations of a water soluble polymer, some molecules willweigh more, some less, than the stated molecular weight). The averagemolecular weight of the polymer is in some aspect between about 5 kDaand about 50 kDa, between about 12 kDa to about 40 kDa or between about20 kDa to about 35 kDa.

In some embodiments, the polymer is modified to have a single reactivegroup, such as an active ester for acylation or an aldehyde foralkylation, so that the degree of polymerization may be controlled. Thepolymer in some embodiments is water soluble so that the protein towhich it is attached does not precipitate in an aqueous environment,such as a physiological environment. In some embodiments, when, forexample, the composition is used for therapeutic use, the polymer ispharmaceutically acceptable. Additionally, in some aspects, the polymeris a mixture of polymers, e.g., a co-polymer, a block co-polymer.

In some embodiments, the polymer is selected from the group consistingof: polyamides, polycarbonates, polyalkylenes and derivatives thereofincluding, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polymers of acrylic and methacrylic esters, includingpoly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate), polyvinyl polymers including polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), andpolyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, celluloses including alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, and cellulosesulphate sodium salt, polypropylene, polyethylenes includingpoly(ethylene glycol), poly(ethylene oxide), and poly(ethyleneterephthalate), and polystyrene.

In some aspects, the polymer is a biodegradable polymer, including asynthetic biodegradable polymer (e.g., polymers of lactic acid andglycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes,poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone)),and a natural biodegradable polymer (e.g., alginate and otherpolysaccharides including dextran and cellulose, collagen, chemicalderivatives thereof (substitutions, additions of chemical groups, forexample, alkyl, alkylene, hydroxylations, oxidations, and othermodifications routinely made by those skilled in the art), albumin andother hydrophilic proteins (e.g., zein and other prolamines andhydrophobic proteins)), as well as any copolymer or mixture thereof. Ingeneral, these materials degrade either by enzymatic hydrolysis orexposure to water in vivo, by surface or bulk erosion.

In some aspects, the polymer is a bioadhesive polymer, such as abioerodible hydrogel described by H. S. Sawhney, C. P. Pathak and J. A.Hubbell in Macromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

In some embodiments, the polymer is a water-soluble polymer or ahydrophilic polymer. Suitable water-soluble polymers are known in theart and include, for example, polyvinylpyrrolidone, hydroxypropylcellulose (HPC; Klucel), hydroxypropyl methylcellulose (HPMC; Methocel),nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropylbutylcellulose, hydroxypropyl pentylcellulose, methyl cellulose,ethylcellulose (Ethocel), hydroxyethyl cellulose, various alkylcelluloses and hydroxyalkyl celluloses, various cellulose ethers,cellulose acetate, carboxymethyl cellulose, sodium carboxymethylcellulose, calcium carboxymethyl cellulose, vinyl acetate/crotonic acidcopolymers, poly-hydroxyalkyl methacrylate, hydroxymethyl methacrylate,methacrylic acid copolymers, polymethacrylic acid,polymethylmethacrylate, maleic anhydride/methyl vinyl ether copolymers,poly vinyl alcohol, sodium and calcium polyacrylic acid, polyacrylicacid, acidic carboxy polymers, carboxypolymethylene, carboxyvinylpolymers, polyoxyethylene polyoxypropylene copolymer,polymethylvinylether co-maleic anhydride, carboxymethylamide, potassiummethacrylate divinylbenzene co-polymer, polyoxyethyleneglycols,polyethylene oxide, and derivatives, salts, and combinations thereof. Insome aspects, the water soluble polymers or mixtures thereof include,but are not limited to, N-linked or O-linked carbohydrates, sugars,phosphates, carbohydrates; sugars; phosphates; polyethylene glycol (PEG)(including the forms of PEG that have been used to derivatize proteins,including mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol);monomethoxy-polyethylene glycol; dextran (such as low molecular weightdextran, of, for example about 6 kD), cellulose; cellulose; othercarbohydrate-based polymers, poly-(N-vinyl pyrrolidone)polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol. Also encompassed by the present invention arebifunctional crosslinking molecules which may be used to preparecovalently attached multimers.

A particularly preferred water-soluble polymer for use herein ispolyethylene glycol (PEG). As used herein, polyethylene glycol is meantto encompass any of the forms of PEG that can be used to derivatizeother proteins, such as mono-(C1-C10) alkoxy- or aryloxy-polyethyleneglycol. PEG is a linear or branched neutral polyether, available in abroad range of molecular weights, and is soluble in water and mostorganic solvents. PEG is effective at excluding other polymers orpeptides when present in water, primarily through its high dynamic chainmobility and hydrophibic nature, thus creating a water shell orhydration sphere when attached to other proteins or polymer surfaces.PEG is nontoxic, non-immunogenic, and approved by the Food and DrugAdministration for internal consumption.

Proteins or enzymes when conjugated to PEG have demonstratedbioactivity, non-antigenic properties, and decreased clearance rateswhen administered in animals. F. M. Veronese et al., Preparation andProperties of Monomethoxypoly(ethylene glycol)-modified Enzymes forTherapeutic Applications, in J. M. Harris ed., Poly(Ethylene Glycol)Chemistry—Biotechnical and Biomedical Applications, 127-36, 1992,incorporated herein by reference. These phenomena are due to theexclusion properties of PEG in preventing recognition by the immunesystem. In addition, PEG has been widely used in surface modificationprocedures to decrease protein adsorption and improve bloodcompatibility. S. W. Kim et al., Ann. N.Y. Acad. Sci. 516: 116-30 1987;Jacobs et al., Artif. Organs 12: 500-501, 1988; Park et al., J. Poly.Sci, Part A 29:1725-31, 1991, incorporated herein by reference.Hydrophobic polymer surfaces, such as polyurethanes and polystyrene canbe modified by the grafting of PEG (MW 3,400) and employed asnonthrombogenic surfaces. Surface properties (contact angle) can be moreconsistent with hydrophilic surfaces, due to the hydrating effect ofPEG. More importantly, protein (albumin and other plasma proteins)adsorption can be greatly reduced, resulting from the high chainmotility, hydration sphere, and protein exclusion properties of PEG.

PEG (MW 3,400) was determined as an optimal size in surfaceimmobilization studies, Park et al., J. Biomed. Mat. Res. 26:739-45,1992, while PEG (MW 5,000) was most beneficial in decreasing proteinantigenicity. (F. M. Veronese et al., In J. M. Harris, et al.,Poly(Ethylene Glycol) Chemistry—Biotechnical and BiomedicalApplications, 127-36.)

Methods for preparing pegylated compounds may comprise the steps of (a)reacting the compound with polyethylene glycol (such as a reactive esteror aldehyde derivative of PEG) under conditions whereby the compoundbecomes attached to one or more PEG groups, and (b) obtaining thereaction product(s). In general, the optimal reaction conditions for theacylation reactions will be determined based on known parameters and thedesired result. For example, the larger the ratio of PEG: compound, thegreater the percentage of poly-pegylated product. In some embodiments,the compound will have a single PEG moiety at the N-terminus. See U.S.Pat. No. 8,234,784, herein incorporated by reference.

In some embodiments, the heterologous moiety is a carbohydrate. In someembodiments, the carbohydrate is a monosaccharide (e.g., glucose,galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose),an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (astarch, amylase, amylopectin, cellulose, chitin, callose, laminarin,xylan, mannan, fucoidan, galactomannan.

In some embodiments, the heterologous moiety is a lipid. The lipid, insome embodiments, is a fatty acid, eicosanoid, prostaglandin,leukotriene, thromboxane, N-acyl ethanolamine), glycerolipid (e.g.,mono-, di-, tri-substituted glycerols), glycerophospholipid (e.g.,phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine,phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterollipid (e.g., steroid, cholesterol), prenol lipid, saccharolipid, or apolyketide, oil, wax, cholesterol, sterol, fat-soluble vitamin,monoglyceride, diglyceride, triglyceride, a phospholipid.

Heterologous Moieties: Therapeutic Agents

In some embodiments, the heterologous moiety is a therapeutic agent. Thetherapeutic agent may be any of those known in the art. Examples oftherapeutic agents that are contemplated herein include, but are notlimited to, natural enzymes, proteins derived from natural sources,recombinant proteins, natural peptides, synthetic peptides, cyclicpeptides, antibodies, receptor agonists, cytotoxic agents,immunoglobins, beta-adrenergic blocking agents, calcium channelblockers, coronary vasodilators, cardiac glycosides, antiarrhythmics,cardiac sympathomemetics, angiotensin converting enzyme (ACE)inhibitors, diuretics, inotropes, cholesterol and triglyceride reducers,bile acid sequestrants, fibrates, 3-hydroxy-3-methylgluteryl (HMG)-CoAreductase inhibitors, niacin derivatives, antiadrenergic agents,alpha-adrenergic blocking agents, centrally acting antiadrenergicagents, vasodilators, potassium-sparing agents, thiazides and relatedagents, angiotensin II receptor antagonists, peripheral vasodilators,antiandrogens, estrogens, antibiotics, retinoids, insulins and analogs,alpha-glucosidase inhibitors, biguanides, meglitinides, sulfonylureas,thizaolidinediones, androgens, progestogens, bone metabolism regulators,anterior pituitary hormones, hypothalamic hormones, posterior pituitaryhormones, gonadotropins, gonadotropin-releasing hormone antagonists,ovulation stimulants, selective estrogen receptor modulators,antithyroid agents, thyroid hormones, bulk forming agents, laxatives,antiperistaltics, flora modifiers, intestinal adsorbents, intestinalanti-infectives, antianorexic, anticachexic, antibulimics, appetitesuppressants, antiobesity agents, antacids, upper gastrointestinal tractagents, anticholinergic agents, aminosalicylic acid derivatives,biological response modifiers, corticosteroids, antispasmodics, 5-HT₄partial agonists, antihistamines, cannabinoids, dopamine antagonists,serotonin antagonists, cytoprotectives, histamine H2-receptorantagonists, mucosal protective agent, proton pump inhibitors, H. pylorieradication therapy, erythropoieses stimulants, hematopoietic agents,anemia agents, heparins, antifibrinolytics, hemostatics, bloodcoagulation factors, adenosine diphosphate inhibitors, glycoproteinreceptor inhibitors, fibrinogen-platelet binding inhibitors,thromboxane-A₂ inhibitors, plasminogen activators, antithromboticagents, glucocorticoids, mineralcorticoids, corticosteroids, selectiveimmunosuppressive agents, antifungals, drugs involved in prophylactictherapy, AIDS-associated infections, cytomegalovirus, non-nucleosidereverse transcriptase inhibitors, nucleoside analog reverse transcriptseinhibitors, protease inhibitors, anemia, Kaposi's sarcoma,aminoglycosides, carbapenems, cephalosporins, glycopoptides,lincosamides, macrolies, oxazolidinones, penicillins, streptogramins,sulfonamides, trimethoprim and derivatives, tetracyclines,anthelmintics, amebicies, biguanides, cinchona alkaloids, folic acidantagonists, quinoline derivatives, Pneumocystis carinii therapy,hydrazides, imidazoles, triazoles, nitroimidzaoles, cyclic amines,neuraminidase inhibitors, nucleosides, phosphate binders, cholinesteraseinhibitors, adjunctive therapy, barbiturates and derivatives,benzodiazepines, gamma aminobutyric acid derivatives, hydantoinderivatives, iminostilbene derivatives, succinimide derivatives,anticonvulsants, ergot alkaloids, antimigrane preparations, biologicalresponse modifiers, carbamic acid eaters, tricyclic derivatives,depolarizing agents, nondepolarizing agents, neuromuscular paralyticagents, CNS stimulants, dopaminergic reagents, monoamine oxidaseinhibitors, COMT inhibitors, alkyl sulphonates, ethylenimines,imidazotetrazines, nitrogen mustard analogs, nitrosoureas,platinum-containing compounds, antimetabolites, purine analogs,pyrimidine analogs, urea derivatives, antracyclines, actinomycinds,camptothecin derivatives, epipodophyllotoxins, taxanes, vinca alkaloidsand analogs, antiandrogens, antiestrogens, nonsteroidal aromataseinhibitors, protein kinase inhibitor antineoplastics,azaspirodecanedione derivatives, anxiolytics, stimulants, monoamindreuptake inhibitors, selective serotonin reuptake inhibitors,antidepressants, benzisooxazole derivatives, butyrophenone derivatives,dibenzodiazepine derivatives, dibenzothiazepine derivatives,diphenylbutylpiperidine derivatives, phenothiazines,thienobenzodiazepine derivatives, thioxanthene derivatives, allergenicextracts, nonsteroidal agents, leukotriene receptor antagonists,xanthines, endothelin receptor antagonist, prostaglandins, lungsurfactants, mucolytics, antimitotics, uricosurics, xanthine oxidaseinhibitors, phosphodiesterase inhibitors, metheamine salts, nitrofuranderivatives, quinolones, smooth muscle relaxants, parasympathomimeticagents, halogenated hydrocarbons, esters of amino benzoic acid, amides(e.g. lidocaine, articaine hydrochloride, bupivacaine hydrochloride),antipyretics, hynotics and sedatives, cyclopyrrolones,pyrazolopyrimidines, nonsteroidal anti-inflammatory drugs, opioids,para-aminophenol derivatives, alcohol dehydrogenase inhibitor, heparinantagonists, adsorbents, emetics, opoid antagonists, cholinesterasereactivators, nicotine replacement therapy, vitamin A analogs andantagonists, vitamin B analogs and antagonists, vitamin C analogs andantagonists, vitamin D analogs and antagonists, vitamin E analogs andantagonists, vitamin K analogs and antagonists.

The compounds of the present disclosures may be conjugated to one ormore cytokines and growth factors that are effective in inhibiting tumormetastasis, and wherein the cytokine or growth factor has been shown tohave an antiproliferative effect on at least one cell population. Suchcytokines, lymphokines, growth factors, or other hematopoietic factorsinclude, but are not limited to: M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF,GM-CSF, thrombopoietin, stem cell factor, and erythropoietin. Additionalgrowth factors for use herein include angiogenin, bone morphogenicprotein-1, bone morphogenic protein-2, bone morphogenic protein-3, bonemorphogenic protein-4, bone morphogenic protein-5, bone morphogenicprotein-6, bone morphogenic protein-7, bone morphogenic protein-8, bonemorphogenic protein-9, bone morphogenic protein-10, bone morphogenicprotein-11, bone morphogenic protein-12, bone morphogenic protein-13,bone morphogenic protein-14, bone morphogenic protein-15, bonemorphogenic protein receptor IA, bone morphogenic protein receptor IB,brain derived neurotrophic factor, ciliary neutrophic factor, ciliaryneutrophic factor receptor α, cytokine-induced neutrophil chemotacticfactor 1, cytokine-induced neutrophil, chemotactic factor 2α,cytokine-induced neutrophil chemotactic factor 2β, β endothelial cellgrowth factor, endothelin 1, epithelial-derived neutrophil attractant,glial cell line-derived neutrophic factor receptor α1, glial cellline-derived neutrophic factor receptor α2, growth related protein,growth related protein α, growth related protein β, growth relatedprotein γ, heparin binding epidermal growth factor, hepatocyte growthfactor, hepatocyte growth factor receptor, insulin-like growth factor I,insulin-like growth factor receptor, insulin-like growth factor II,insulin-like growth factor binding protein, keratinocyte growth factor,leukemia inhibitory factor, leukemia inhibitory factor receptor α, nervegrowth factor nerve growth factor receptor, neurotrophin-3,neurotrophin-4, pre-B cell growth stimulating factor, stem cell factor,stem cell factor receptor, transforming growth factor α, transforminggrowth factor β, transforming growth factor β1, transforming growthfactor β1.2, transforming growth factor β2, transforming growth factorβ3, transforming growth factor β5, latent transforming growth factor β1,transforming growth factor β binding protein I, transforming growthfactor β binding protein II, transforming growth factor β bindingprotein III, tumor necrosis factor receptor type I, tumor necrosisfactor receptor type II, urokinase-type plasminogen activator receptor,and chimeric proteins and biologically or immunologically activefragments thereof.

In some embodiments, the conjugate comprises a compound as describedherein and a cytotoxic agent. The cytotoxic agent is any molecule(chemical or biochemical) which is toxic to a cell. In some aspects,when a cytotoxic agent is conjugated to a compound of the presentdisclosures, the results obtained are synergistic. That is to say, theeffectiveness of the combination therapy of a compound and the cytotoxicagent is synergistic, i.e., the effectiveness is greater than theeffectiveness expected from the additive individual effects of each.Therefore, the dosage of the cytotoxic agent can be reduced and thus,the risk of the toxicity problems and other side effects isconcomitantly reduced. In some embodiments, the cytotoxic agent is achemotherapeutic agent. Chemotherapeutic agents are known in the art andinclude, but not limited to, platinum coordination compounds,topoisomerase inhibitors, antibiotics, antimitotic alkaloids anddifluoronucleosides, as described in U.S. Pat. No. 6,630,124.

In some embodiments, the chemotherapeutic agent is a platinumcoordination compound. The term “platinum coordination compound” refersto any tumor cell growth inhibiting platinum coordination compound thatprovides the platinum in the form of an ion. In some embodiments, theplatinum coordination compound is cis-diamminediaquoplatinum (II)-ion;chloro(diethylenetriamine)-platinum(II)chloride;dichloro(ethylenediamine)-platinum(II),diammine(1,1-cyclobutanedicarboxylato) platinum(II) (carboplatin);spiroplatin; iproplatin; diammine(2-ethylmalonato)-platinum(II);ethylenediaminemalonatoplatinum(II);aqua(1,2-diaminodyclohexane)-sulfatoplatinum(II);(1,2-diaminocyclohexane)malonatoplatinum(II);(4-caroxyphthalato)(1,2-diaminocyclohexane)platinum(II);(1,2-diaminocyclohexane)-(isocitrato)platinum(II);(1,2-diaminocyclohexane)cis(pyruvato)platinum(II);(1,2-diaminocyclohexane)oxalatoplatinum(II); ormaplatin; andtetraplatin.

In some embodiments, cisplatin is the platinum coordination compoundemployed in the compositions and methods of the present invention.Cisplatin is commercially available under the name PLATINOL™ fromBristol Myers-Squibb Corporation and is available as a powder forconstitution with water, sterile saline or other suitable vehicle. Otherplatinum coordination compounds suitable for use in the presentinvention are known and are available commercially and/or can beprepared by conventional techniques. Cisplatin, orcis-dichlorodiaminoplatinum II, has been used successfully for manyyears as a chemotherapeutic agent in the treatment of various humansolid malignant tumors. More recently, other diamino-platinum complexeshave also shown efficacy as chemotherapeutic agents in the treatment ofvarious human solid malignant tumors. Such diamino-platinum complexesinclude, but are not limited to, spiroplatinum and carboplatinum.Although cisplatin and other diamino-platinum complexes have been widelyused as chemotherapeutic agents in humans, they have had to be deliveredat high dosage levels that can lead to toxicity problems such as kidneydamage.

In some embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerases are enzymes that are capable of altering DNAtopology in eukaryotic cells. They are critical for cellular functionsand cell proliferation. Generally, there are two classes oftopoisomerases in eukaryotic cells, type I and type II. Topoisomerase Iis a monomeric enzyme of approximately 100,000 molecular weight. Theenzyme binds to DNA and introduces a transient single-strand break,unwinds the double helix (or allows it to unwind), and subsequentlyreseals the break before dissociating from the DNA strand. Varioustopoisomerase inhibitors have recently shown clinical efficacy in thetreatment of humans afflicted with ovarian, cancer, esophageal cancer ornon-small cell lung carcinoma.

In some aspects, the topoisomerase inhibitor is camptothecin or acamptothecin analog. Camptothecin is a water-insoluble, cytotoxicalkaloid produced by Camptotheca accuminata trees indigenous to Chinaand Nothapodytes foetida trees indigenous to India. Camptothecinexhibits tumor cell growth inhibiting activity against a number of tumorcells. Compounds of the camptothecin analog class are typically specificinhibitors of DNA topoisomerase I. By the term “inhibitor oftopoisomerase” is meant any tumor cell growth inhibiting compound thatis structurally related to camptothecin. Compounds of the camptothecinanalog class include, but are not limited to; topotecan, irinotecan and9-aminocamptothecin.

In additional embodiments, the cytotoxic agent is any tumor cell growthinhibiting camptothecin analog claimed or described in: U.S. Pat. No.5,004,758, issued on Apr. 2, 1991 and European Patent Application Number88311366.4, published on Jun. 21, 1989 as 20′ Publication Number EP 0321 122; U.S. Pat. No. 4,604,463, issued on Aug. 5, 1986 and EuropeanPatent Application Publication Number EP 0 137 145, published on Apr.17, 1985; U.S. Pat. No. 4,473,692, issued on Sep. 25, 1984 and EuropeanPatent Application Publication Number EP 0 074 256, published on Mar.16, 1983; U.S. Pat. No. 4,545,880, issued on Oct. 8, 1985 and EuropeanPatent Application Publication Number EP 0 074 256, published on Mar.16, 1983; European Patent Application Publication Number EP 0 088 642,published on Sep. 14, 1983; Wani et al., J. Med. Chem., 29, 2358-2363(1986); Nitta et al., Proc. 14th International Congr. Chemotherapy,Kyoto, 1985, Tokyo Press, Anticancer Section 1, p. 28-30, especially acompound called CPT-11. CPT-11 is a camptothecin analog with a4-(piperidino)-piperidine side chain joined through a carbamate linkageat C-10 of 10-hydroxy-7-ethyl camptothecin. CPT-11 is currentlyundergoing human clinical trials and is also referred to as irinotecan;Wani et al, J. Med. Chem., 23, 554 (1980); Wani et. al., J. Med. Chem.,30, 1774 (1987); U.S. Pat. No. 4,342,776, issued on Aug. 3, 1982; U.S.patent application Ser. No. 581,916, filed on Sep. 13, 1990 and EuropeanPatent Application Publication Number EP 418 099, published on Mar. 20,1991; U.S. Pat. No. 4,513,138, issued on Apr. 23, 1985 and EuropeanPatent Application Publication Number EP 0 074 770, published on Mar.23, 1983; U.S. Pat. No. 4,399,276, issued on Aug. 16, 1983 and EuropeanPatent Application Publication Number 0 056 692, published on Jul. 28,1982; the entire disclosure of each of which is hereby incorporated byreference. All of the above-listed compounds of the camptothecin analogclass are available commercially and/or can be prepared by conventionaltechniques including those described in the above-listed references. Thetopoisomerase inhibitor may be selected from the group consisting oftopotecan, irinotecan and 9-aminocamptothecin.

The preparation of numerous compounds of the camptothecin analog class(including pharmaceutically acceptable salts, hydrates and solvatesthereof) as well as the preparation of oral and parenteralpharmaceutical compositions comprising such a compounds of thecamptothecin analog class and an inert, pharmaceutically acceptablecarrier or diluent, is extensively described in U.S. Pat. No. 5,004,758,issued on Apr. 2, 1991 and European Patent Application Number88311366.4, published on Jun. 21, 1989 as Publication Number EP 0 321122, the teachings of which are incorporated herein by reference.

In still yet other embodiments of the present disclosures, thechemotherapeutic agent is an antibiotic compound. Suitable antibioticinclude, but are not limited to, doxorubicin, mitomycin, bleomycin,daunorubicin and streptozocin.

In some embodiments, the chemotherapeutic agent is an antimitoticalkaloid. In general, antimitotic alkaloids can be extracted fromCantharanthus roseus, and have been shown to be efficacious asanticancer chemotherapy agents. A great number of semi-syntheticderivatives have been studied both chemically and pharmacologically(see, O. Van Tellingen et al, Anticancer Research, 12, 1699-1716(1992)). The antimitotic alkaloids of the present invention include, butare not limited to, vinblastine, vincristine, vindesine, Taxol andvinorelbine. The latter two antimitotic alkaloids are commerciallyavailable from Eli Lilly and Company, and Pierre Fabre Laboratories,respectively (see, U.S. Pat. No. 5,620,985). In a preferred aspect ofthe present invention, the antimitotic alkaloid is vinorelbine.

In other embodiments of the present disclosures, the chemotherapeuticagent is a difluoronucleoside. 2′-deoxy-2′,2′-difluoronucleosides areknown in the art as having antiviral activity. Such compounds aredisclosed and taught in U.S. Pat. Nos. 4,526,988 and 4,808614. EuropeanPatent Application Publication 184,365 discloses that these samedifluoronucleosides have oncolytic activity. In certain specificaspects, the 2′-deoxy-2′,2′-difluoronucleoside used in the compositionsand methods of the present invention is 2′-deoxy-2′,2′-difluorocytidinehydrochloride, also known as gemcitabine hydrochloride. Gemcitabine iscommercially available or can be synthesized in a multi-step process asdisclosed and taught in U.S. Pat. Nos. 4,526,988, 4,808,614 and5,223,608, the teachings of which are incorporated herein by reference.

Conjugates: Targeted Forms

One of ordinary skill in the art will readily appreciate that thecompounds of the present disclosure can be modified in any number ofways, such that the therapeutic or prophylactic efficacy of the compoundof the present disclosures is increased through the modification. Forinstance, the compound of the present disclosure can be conjugatedeither directly or indirectly through a linker to a targeting moiety.The practice of conjugating compounds to targeting moieties is known inthe art. See, for instance, Wadhwa et al., J Drug Targeting, 3, 111-127(1995) and U.S. Pat. No. 5,087,616. The term “targeting moiety” as usedherein, refers to any molecule or agent that specifically recognizes andbinds to a cell-surface receptor, such that the targeting moiety directsthe delivery of the compound of the present disclosures to a populationof cells on which surface the receptor is expressed. Targeting moietiesinclude, but are not limited to, antibodies, or fragments thereof,peptides, hormones, growth factors, cytokines, and any other natural ornon-natural ligands, which bind to cell surface receptors (e.g.,Epithelial Growth Factor Receptor (EGFR), T-cell receptor (TCR), B-cellreceptor (BCR), CD28, Platelet-derived Growth Factor Receptor (PDGF),nicotinic acetylcholine receptor (nAChR), etc.). As used herein a“linker” is a bond, molecule or group of molecules that binds twoseparate entities to one another. Linkers may provide for optimalspacing of the two entities or may further supply a labile linkage thatallows the two entities to be separated from each other. Labile linkagesinclude photocleavable groups, acid-labile moieties, base-labilemoieties and enzyme-cleavable groups. The term “linker” in someembodiments refers to any agent or molecule that bridges the compound ofthe present disclosures to the targeting moiety. The linker may be anyof those described herein under the section entitled “Linkers.” One ofordinary skill in the art recognizes that sites on the compound of thepresent disclosures, which are not necessary for the function of thecompound, are ideal sites for attaching a linker and/or a targetingmoiety, provided that the linker and/or targeting moiety, once attachedto the compound, do(es) not interfere with the function of the compound,i.e., the ability to inhibit the binding interaction between EGFR andHSP90, as described herein.

Linkers

In some embodiments, the conjugate comprises a linker that joins thecompound of the present disclosures to the heterologous moiety. In someaspects, the linker comprises a chain of atoms from 1 to about 60, or 1to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10to 20 atoms long. In some embodiments, the chain atoms are all carbonatoms. In some embodiments, the chain atoms in the backbone of thelinker are selected from the group consisting of C, O, N, and S. Chainatoms and linkers may be selected according to their expected solubility(hydrophilicity) so as to provide a more soluble conjugate. In someembodiments, the linker provides a functional group that is subject tocleavage by an enzyme or other catalyst or hydrolytic conditions foundin the target tissue or organ or cell. In some embodiments, the lengthof the linker is long enough to reduce the potential for sterichindrance. In some embodiments, the linker is an amino acid or apeptidyl linker. Such peptidyl linkers may be any length. Exemplarylinkers are from about 1 to 50 amino acids in length, 5 to 50, 3 to 5, 5to 10, 5 to 15, or 10 to 30 amino acids in length.

Dimers & Multimers

In some embodiments, the compound is provided as a dimer or a multimerin which more than one compound of the present disclosures are linkedtogether. The dimer in some aspects is a homodimer comprising twocompounds of the same type (e.g., same structure) linked together. Inalternative aspects, the dimer is a heterodimer comprising two compoundsof the present disclosures, wherein the two compounds are structurallydistinct from each other. The multimer in some aspects is a homomultimercomprising more than one compound of the present disclosures and eachcompound are of the same type (e.g., same structure). In alternativeaspects, the multimer is a heteromultimer comprising more than onecompound of the present disclosures and wherein at least two compoundsof the heteromultimer are structurally distinct from the other. Two ormore of the compounds can be linked together using standard linkingagents and procedures known to those skilled in the art. In certainembodiments, the linker connecting the two (or more) compounds is alinker as described in the section entitled “Linkers.” In someembodiments, the linker is a disulfide bond. For example, each monomerof the dimer may comprise a sulfhydryl and the sulfur atom of eachparticipates in the formation of the disulfide bond.

Compositions

The present disclosures further provide compositions comprising acompound that inhibits a binding interaction between EGFR and HSP90,e.g., an antibody, antigen binding fragment, aptamer, peptide, peptideanalog, pharmaceutically acceptable salt, conjugate, multimer, dimer, asdescribed herein. The compositions in some aspects comprise thecompounds in isolated and/or purified form. In some aspects, thecomposition comprises a single type (e.g., structure) of a compound ofthe present disclosures or comprises a combination of two or morecompounds of the present disclosures, wherein the combination comprisestwo or more compounds of different types (e.g., structures).

In some aspects, the composition comprises agents which enhance thechemico-physico features of the compound, e.g., via stabilizing thecompound at certain temperatures, e.g., room temperature, increasingshelf life, reducing degradation, e.g., oxidation protease mediateddegradation, increasing half-life of the compound, etc. In some aspects,the composition comprises any of the agents disclosed herein as aheterologous moiety or conjugate moiety, optionally in admixture withthe compounds of the present disclosures or conjugated to the compounds.

In certain aspects, the composition comprises a delivery agent whichaids in localizing the compound of the present disclosures to theappropriate place. In certain aspects, the composition comprises acompound of the present disclosures (e.g., a peptide that inhibits abinding interaction between EGFR and HSP90) and a peptide deliveryagent. In some aspects, the peptide delivery agent is a cell penetratingpeptide (CPP). In particular aspects, the composition comprises a CPPfused to the compound, e.g., the composition comprises a fusion peptideproduct comprising a peptide of the present disclosures that inhibits abinding interaction between EGFR and HSP90 fused to a CPP. In certainaspects, the CPP comprises the amino acid sequence YGRKKRRQRRR (SEQ IDNO: 31).

Pharmaceutical Compositions and Formulations

In yet other aspects of the present disclosures, the compositioncomprises a compound that inhibits a binding interaction between EGFRand HSP90 and additionally comprises a pharmaceutically acceptablecarrier, diluents, or excipient. In some embodiments, the compound ofthe present disclosures, the pharmaceutically acceptable salt, theconjugate, the dimer or multimer, of the present disclosures(hereinafter referred to as “active agents”) is formulated into apharmaceutical composition comprising the active agent, along with apharmaceutically acceptable carrier, diluent, or excipient. In thisregard, the present disclosures further provides pharmaceuticalcompositions comprising an active agent that inhibits a bindinginteraction between EGFR and HSP90 which is intended for administrationto a subject, e.g., a mammal.

In some embodiments, the active agent is present in the pharmaceuticalcomposition at a purity level suitable for administration to a patient.In some embodiments, the active agent has a purity level of at leastabout 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98% or about 99%, and a pharmaceuticallyacceptable diluent, carrier or excipient. The pharmaceutical compositionin some aspects comprises the active agent of the present disclosure ata concentration of at least A, wherein A is about 0.001 mg/ml, about0.01 mg/ml, 0 about 1 mg/ml, about 0.5 mg/ml, about 1 mg/ml, about 2mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 11 mg/ml,about 12 mg/ml, about 13 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16mg/ml, about 17 mg/ml, about 18 mg/ml, about 19 mg/ml, about 20 mg/ml,about 21 mg/ml, about 22 mg/ml, about 23 mg/ml, about 24 mg/ml, about 25mg/ml or higher. In some embodiments, the pharmaceutical compositioncomprises the active agent at a concentration of at most B, wherein B isabout 30 mg/ml, about 25 mg/ml, about 24 mg/ml, about 23, mg/ml, about22 mg/ml, about 21 mg/ml, about 20 mg/ml, about 19 mg/ml, about 18mg/ml, about 17 mg/ml, about 16 mg/ml, about 15 mg/ml, about 14 mg/ml,about 13 mg/ml, about 12 mg/ml, about 11 mg/ml, about 10 mg/ml, about 9mg/ml, about 8 mg/ml, about 7 mg/ml, about 6 mg/ml, about 5 mg/ml, about4 mg/ml, about 3 mg/ml, about 2 mg/ml, about 1 mg/ml, or about 0.1mg/ml. In some embodiments, the compositions may contain an active agentat a concentration range of A to B mg/ml, for example, about 0.001 toabout 30.0 mg/ml.

Depending on the route of administration, the particular active agentintended for use, as well as other factors, the pharmaceuticalcomposition may comprise additional pharmaceutically acceptableingredients, including, for example, acidifying agents, additives,adsorbents, aerosol propellants, air displacement agents, alkalizingagents, anticaking agents, anticoagulants, antimicrobial preservatives,antioxidants, antiseptics, bases, binders, buffering agents, chelatingagents, coating agents, coloring agents, desiccants, detergents,diluents, disinfectants, disintegrants, dispersing agents, dissolutionenhancing agents, dyes, emollients, emulsifying agents, emulsionstabilizers, fillers, film forming agents, flavor enhancers, flavoringagents, flow enhancers, gelling agents, granulating agents, humectants,lubricants, mucoadhesives, ointment bases, ointments, oleaginousvehicles, organic bases, pastille bases, pigments, plasticizers,polishing agents, preservatives, sequestering agents, skin penetrants,solubilizing agents, solvents, stabilizing agents, suppository bases,surface active agents, surfactants, suspending agents, sweeteningagents, therapeutic agents, thickening agents, tonicity agents, toxicityagents, viscosity-increasing agents, water-absorbing agents,water-miscible cosolvents, water softeners, or wetting agents.

Accordingly, in some embodiments, the pharmaceutical compositioncomprises any one or a combination of the following components: acacia,acesulfame potassium, acetyltributyl citrate, acetyltriethyl citrate,agar, albumin, alcohol, dehydrated alcohol, denatured alcohol, dilutealcohol, aleuritic acid, alginic acid, aliphatic polyesters, alumina,aluminum hydroxide, aluminum stearate, amylopectin, α-amylose, ascorbicacid, ascorbyl palmitate, aspartame, bacteriostatic water for injection,bentonite, bentonite magma, benzalkonium chloride, benzethoniumchloride, benzoic acid, benzyl alcohol, benzyl benzoate, bronopol,butylated hydroxyanisole, butylated hydroxytoluene, butylparaben,butylparaben sodium, calcium alginate, calcium ascorbate, calciumcarbonate, calcium cyclamate, dibasic anhydrous calcium phosphate,dibasic dehydrate calcium phosphate, tribasic calcium phosphate, calciumpropionate, calcium silicate, calcium sorbate, calcium stearate, calciumsulfate, calcium sulfate hemihydrate, canola oil, carbomer, carbondioxide, carboxymethyl cellulose calcium, carboxymethyl cellulosesodium, β-carotene, carrageenan, castor oil, hydrogenated castor oil,cationic emulsifying wax, cellulose acetate, cellulose acetatephthalate, ethyl cellulose, microcrystalline cellulose, powderedcellulose, silicified microcrystalline cellulose, sodium carboxymethylcellulose, cetostearyl alcohol, cetrimide, cetyl alcohol, chlorhexidine,chlorobutanol, chlorocresol, cholesterol, chlorhexidine acetate,chlorhexidine gluconate, chlorhexidine hydrochloride,chlorodifluoroethane (HCFC), chlorodifluoromethane, chlorofluorocarbons(CFC) chlorophenoxyethanol, chloroxylenol, corn syrup solids, anhydrouscitric acid, citric acid monohydrate, cocoa butter, coloring agents,corn oil, cottonseed oil, cresol, m-cresol, o-cresol, p-cresol,croscarmellose sodium, crospovidone, cyclamic acid, cyclodextrins,dextrates, dextrin, dextrose, dextrose anhydrous, diazolidinyl urea,dibutyl phthalate, dibutyl sebacate, diethanolamine, diethyl phthalate,difluoroethane (HFC), dimethyl-β-cyclodextrin, cyclodextrin-typecompounds such as Captisol®, dimethyl ether, dimethyl phthalate,dipotassium edentate, disodium edentate, disodium hydrogen phosphate,docusate calcium, docusate potassium, docusate sodium, dodecyl gallate,dodecyltrimethylammonium bromide, edentate calcium disodium, edtic acid,eglumine, ethyl alcohol, ethylcellulose, ethyl gallate, ethyl laurate,ethyl maltol, ethyl oleate, ethylparaben, ethylparaben potassium,ethylparaben sodium, ethyl vanillin, fructose, fructose liquid, fructosemilled, fructose pyrogen-free, powdered fructose, fumaric acid, gelatin,glucose, liquid glucose, glyceride mixtures of saturated vegetable fattyacids, glycerin, glyceryl behenate, glyceryl monooleate, glycerylmonostearate, self-emulsifying glyceryl monostearate, glycerylpalmitostearate, glycine, glycols, glycofurol, guar gum,heptafluoropropane (HFC), hexadecyltrimethylammonium bromide, highfructose syrup, human serum albumin, hydrocarbons (HC), dilutehydrochloric acid, hydrogenated vegetable oil, type II, hydroxyethylcellulose, 2-hydroxyethyl-β-cyclodextrin, hydroxypropyl cellulose,low-substituted hydroxypropyl cellulose, 2-hydroxypropyl-β-cyclodextrin,hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate,imidurea, indigo carmine, ion exchangers, iron oxides, isopropylalcohol, isopropyl myristate, isopropyl palmitate, isotonic saline,kaolin, lactic acid, lactitol, lactose, lanolin, lanolin alcohols,anhydrous lanolin, lecithin, magnesium aluminum silicate, magnesiumcarbonate, normal magnesium carbonate, magnesium carbonate anhydrous,magnesium carbonate hydroxide, magnesium hydroxide, magnesium laurylsulfate, magnesium oxide, magnesium silicate, magnesium stearate,magnesium trisilicate, magnesium trisilicate anhydrous, malic acid,malt, maltitol, maltitol solution, maltodextrin, maltol, maltose,mannitol, medium chain triglycerides, meglumine, menthol,methylcellulose, methyl methacrylate, methyl oleate, methylparaben,methylparaben potassium, methylparaben sodium, microcrystallinecellulose and carboxymethylcellulose sodium, mineral oil, light mineraloil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine,montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin,peanut oil, petrolatum, petrolatum and lanolin alcohols, pharmaceuticalglaze, phenol, liquified phenol, phenoxyethanol, phenoxypropanol,phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate,phenylmercuric nitrate, polacrilin, polacrilin potassium, poloxamer,polydextrose, polyethylene glycol, polyethylene oxide, polyacrylates,polyethylene-polyoxypropylene-block polymers, polymethacrylates,polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives,polyoxyethylene sorbitol fatty acid esters, polyoxyethylene stearates,polyvinyl alcohol, polyvinyl pyrrolidone, potassium alginate, potassiumbenzoate, potassium bicarbonate, potassium bisulfite, potassiumchloride, postassium citrate, potassium citrate anhydrous, potassiumhydrogen phosphate, potassium metabisulfite, monobasic potassiumphosphate, potassium propionate, potassium sorbate, povidone, propanol,propionic acid, propylene carbonate, propylene glycol, propylene glycolalginate, propyl gallate, propylparaben, propylparaben potassium,propylparaben sodium, protamine sulfate, rapeseed oil, Ringer'ssolution, saccharin, saccharin ammonium, saccharin calcium, saccharinsodium, safflower oil, saponite, serum proteins, sesame oil, colloidalsilica, colloidal silicon dioxide, sodium alginate, sodium ascorbate,sodium benzoate, sodium bicarbonate, sodium bisulfite, sodium chloride,anhydrous sodium citrate, sodium citrate dehydrate, sodium chloride,sodium cyclamate, sodium edentate, sodium dodecyl sulfate, sodium laurylsulfate, sodium metabisulfite, sodium phosphate, dibasic, sodiumphosphate, monobasic, sodium phosphate, tribasic, anhydrous sodiumpropionate, sodium propionate, sodium sorbate, sodium starch glycolate,sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters(sorbitan fatty esters), sorbitol, sorbitol solution 70%, soybean oil,spermaceti wax, starch, corn starch, potato starch, pregelatinizedstarch, sterilizable maize starch, stearic acid, purified stearic acid,stearyl alcohol, sucrose, sugars, compressible sugar, confectioner'ssugar, sugar spheres, invert sugar, Sugartab, Sunset Yellow FCF,synthetic paraffin, talc, tartaric acid, tartrazine, tetrafluoroethane(HFC), theobroma oil, thimerosal, titanium dioxide, alpha tocopherol,tocopheryl acetate, alpha tocopheryl acid succinate, beta-tocopherol,delta-tocopherol, gamma-tocopherol, tragacanth, triacetin, tributylcitrate, triethanolamine, triethyl citrate, trimethyl-β-cyclodextrin,trimethyltetradecylammonium bromide, tris buffer, trisodium edentate,vanillin, type I hydrogenated vegetable oil, water, soft water, hardwater, carbon dioxide-free water, pyrogen-free water, water forinjection, sterile water for inhalation, sterile water for injection,sterile water for irrigation, waxes, anionic emulsifying wax, carnaubawax, cationic emulsifying wax, cetyl ester wax, microcrystalline wax,nonionic emulsifying wax, suppository wax, white wax, yellow wax, whitepetrolatum, wool fat, xanthan gum, xylitol, zein, zinc propionate, zincsalts, zinc stearate, or any excipient in the Handbook of PharmaceuticalExcipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London,UK, 2000), which is incorporated by reference in its entirety.Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin(Mack Publishing Co., Easton, Pa., 1980), which is incorporated byreference in its entirety, discloses various components used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional agent is incompatible with the pharmaceutical compositions,its use in pharmaceutical compositions is contemplated. Supplementaryactive ingredients also can be incorporated into the compositions.

In some embodiments, the foregoing component(s) may be present in thepharmaceutical composition at any concentration, such as, for example,at least A, wherein A is 0.0001% w/v, 0.001% w/v, 0.01% w/v, 0.1% w/v,1% w/v, 2% w/v, 5% w/v, 10% w/v, 20% w/v, 30% w/v, 40% w/v, 50% w/v, 60%w/v, 70% w/v, 80% w/v, or 90% w/v. In some embodiments, the foregoingcomponent(s) may be present in the pharmaceutical composition at anyconcentration, such as, for example, at most B, wherein B is 90% w/v,80% w/v, 70% w/v, 60% w/v, 50% w/v, 40% w/v, 30% w/v, 20% w/v, 10% w/v,5% w/v, 2% w/v, 1% w/v, 0.1% w/v, 0.001% w/v, or 0.0001%. In otherembodiments, the foregoing component(s) may be present in thepharmaceutical composition at any concentration range, such as, forexample from about A to about B. In some embodiments, A is 0.0001% and Bis 90%.

The pharmaceutical compositions may be formulated to achieve aphysiologically compatible pH. In some embodiments, the pH of thepharmaceutical composition may be at least 5, at least 5.5, at least 6,at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, atleast 9, at least 9.5, at least 10, or at least 10.5 up to and includingpH 11, depending on the formulation and route of administration. Incertain embodiments, the pharmaceutical compositions may comprisebuffering agents to achieve a physiological compatible pH. The bufferingagents may include any compounds capable of buffering at the desired pHsuch as, for example, phosphate buffers (e.g., PBS), triethanolamine,Tris, bicine, TAPS, tricine, HEPES, TES, MOPS, PIPES, cacodylate, MES,and others. In certain embodiments, the strength of the buffer is atleast 0.5 mM, at least 1 mM, at least 5 mM, at least 10 mM, at least 20mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, atleast 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least120 mM, at least 150 mM, or at least 200 mM. In some embodiments, thestrength of the buffer is no more than 300 mM (e.g., at most 200 mM, atmost 100 mM, at most 90 mM, at most 80 mM, at most 70 mM, at most 60 mM,at most 50 mM, at most 40 mM, at most 30 mM, at most 20 mM, at most 10mM, at most 5 mM, at most 1 mM).

Routes of Administration

With regard to the present disclosures, the active agent, pharmaceuticalcomposition comprising the same, may be administered to the subject viaany suitable route of administration. The following discussion on routesof administration is merely provided to illustrate exemplary embodimentsand should not be construed as limiting the scope in any way.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the active agent of thepresent disclosure dissolved in diluents, such as water, saline, ororange juice; (b) capsules, sachets, tablets, lozenges, and troches,each containing a predetermined amount of the active ingredient, assolids or granules; (c) powders; (d) suspensions in an appropriateliquid; and (e) suitable emulsions. Liquid formulations may includediluents, such as water and alcohols, for example, ethanol, benzylalcohol, and the polyethylene alcohols, either with or without theaddition of a pharmaceutically acceptable surfactant. Capsule forms canbe of the ordinary hard- or soft-shelled gelatin type containing, forexample, surfactants, lubricants, and inert fillers, such as lactose,sucrose, calcium phosphate, and corn starch. Tablet forms can includeone or more of lactose, sucrose, mannitol, corn starch, potato starch,alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, calcium stearate, zinc stearate, stearic acid, and otherexcipients, colorants, diluents, buffering agents, disintegratingagents, moistening agents, preservatives, flavoring agents, and otherpharmacologically compatible excipients. Lozenge forms can comprise theactive agent of the present disclosure in a flavor, usually sucrose andacacia or tragacanth, as well as pastilles comprising the active agentof the present disclosure in an inert base, such as gelatin andglycerin, or sucrose and acacia, emulsions, gels, and the likecontaining, in addition to, such excipients as are known in the art.

The active agents of the present disclosure, alone or in combinationwith other suitable components, can be delivered via pulmonaryadministration and can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer. Such spray formulations also may be usedto spray mucosa. In some embodiments, the active agent is formulatedinto a powder blend or into microparticles or nanoparticles. Suitablepulmonary formulations are known in the art. See, e.g., Qian et al., IntJ Pharm 366: 218-220 (2009); Adjei and Garren, Pharmaceutical Research,7(6): 565-569 (1990); Kawashima et al., J Controlled Release 62(1-2):279-287 (1999); Liu et al., Pharm Res 10(2): 228-232 (1993);International Patent Application Publication Nos. WO 2007/133747 and WO2007/141411.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The term, “parenteral” means not through the alimentary canal but bysome other route such as subcutaneous, intramuscular, intraspinal, orintravenous. The active agent of the present disclosure can beadministered with a physiologically acceptable diluent in apharmaceutical carrier, such as a sterile liquid or mixture of liquids,including water, saline, aqueous dextrose and related sugar solutions,an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such aspropylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol,ketals such as 2,2-dimethyl-153-dioxolane-4-methanol, ethers,poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters orglycerides, or acetylated fatty acid glycerides with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations in some embodiments contain from about 0.5%to about 25% by weight of the active agent of the present disclosure insolution. Preservatives and buffers may be used. In order to minimize oreliminate irritation at the site of injection, such compositions maycontain one or more nonionic surfactants having a hydrophile-lipophilebalance (HLB) of from about 12 to about 17. The quantity of surfactantin such formulations will typically range from about 5% to about 15% byweight. Suitable surfactants include polyethylene glycol sorbitan fattyacid esters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations in some aspects are presented in unit-dose or multi-dosesealed containers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions in some aspects are prepared from sterile powders, granules,and tablets of the kind previously described.

Injectable formulations are in accordance with the invention. Therequirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986)).

Additionally, the active agent of the present disclosures can be madeinto suppositories for rectal administration by mixing with a variety ofbases, such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration can be presented as pessaries,tampons, creams, gels, pastes, foams, or spray formulas containing, inaddition to the active ingredient, such carriers as are known in the artto be appropriate.

It will be appreciated by one of skill in the art that, in addition tothe above-described pharmaceutical compositions, the active agent of thedisclosure can be formulated as inclusion complexes, such ascyclodextrin inclusion complexes, or liposomes.

Dosages

The active agents of the disclosure are believed to be useful in methodsof inhibiting a binding interaction between EGFR and HSP90, methods ofincreasing EGFR degradation, methods of treating cancer in a subject,and methods of sensitizing tumors to treatment, as further describedherein. For purposes of the disclosure, the amount or dose of the activeagent administered should be sufficient to effect, e.g., a therapeuticor prophylactic response, in the subject or animal over a reasonabletime frame. For example, the dose of the active agent of the presentdisclosure should be sufficient to treat cancer as described herein in aperiod of from about 1 to 4 minutes, 1 to 4 hours or 1 to 4 weeks orlonger, e.g., 5 to 20 or more weeks, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular active agent and thecondition of the animal (e.g., human), as well as the body weight of theanimal (e.g., human) to be treated.

Many assays for determining an administered dose are known in the art.For purposes herein, an assay, which comprises comparing the extent towhich cancer is treated upon administration of a given dose of theactive agent of the present disclosure to a mammal among a set ofmammals, each set of which is given a different dose of the activeagent, could be used to determine a starting dose to be administered toa mammal. The extent to which cancer is treated upon administration of acertain dose can be represented by, for example, the cytotoxicity of theactive agent or the extent of tumor regression achieved with the activeagent in a mouse xenograft model. Methods of measuring cytotoxicity ofcompounds and methods of assaying tumor regression are known in the art,including, for instance, the methods described in the EXAMPLES set forthbelow.

The dose of the active agent of the present disclosure also will bedetermined by the existence, nature and extent of any adverse sideeffects that might accompany the administration of a particular activeagent of the present disclosure. Typically, the attending physician willdecide the dosage of the active agent of the present disclosure withwhich to treat each individual patient, taking into consideration avariety of factors, such as age, body weight, general health, diet, sex,active agent of the present disclosure to be administered, route ofadministration, and the severity of the condition being treated. By wayof example and not intending to limit the invention, the dose of theactive agent of the present disclosure can be about 0.0001 to about 1g/kg body weight of the subject being treated/day, from about 0.0001 toabout 0.001 g/kg body weight/day, or about 0.01 mg to about 1 g/kg bodyweight/day. In some embodiments, the dose is up to 50 mg/kg body weight,from about 5 to about 30 mg/kg body weight or from about 8 to about 10mg/kg body weight.

Controlled Release Formulations

In some embodiments, the active agents described herein can be modifiedinto a depot form, such that the manner in which the active agent of thepresent disclosures is released into the body to which it isadministered is controlled with respect to time and location within thebody (see, for example, U.S. Pat. No. 4,450,150). Depot forms of activeagents of the present disclosures can be, for example, an implantablecomposition comprising the active agents and a porous or non-porousmaterial, such as a polymer, wherein the active agent is encapsulated byor diffused throughout the material and/or degradation of the non-porousmaterial. The depot is then implanted into the desired location withinthe body of the subject and the active agent is released from theimplant at a predetermined rate.

The pharmaceutical composition comprising the active agent in certainaspects is modified to have any type of in vivo release profile. In someaspects, the pharmaceutical composition is an immediate release,controlled release, sustained release, extended release, delayedrelease, or bi-phasic release formulation. Methods of formulatingpeptides for controlled release are known in the art. See, for example,Qian et al., J Pharm 374: 46-52 (2009) and International PatentApplication Publication Nos. WO 2008/130158, WO2004/033036;WO2000/032218; and WO 1999/040942.

The instant compositions may further comprise, for example, micelles orliposomes, or some other encapsulated form, or may be administered in anextended release form to provide a prolonged storage and/or deliveryeffect.

Timing of Administration

The disclosed pharmaceutical compositions and formulations may beadministered according to any regimen including, for example, daily (1time per day, 2 times per day, 3 times per day, 4 times per day, 5 timesper day, 6 times per day), every two days, every three days, every fourdays, every five days, every six days, weekly, bi-weekly, every threeweeks, monthly, or bi-monthly. Timing, like dosing can be fine-tunedbased on dose-response studies, efficacy, and toxicity data, andinitially guaged based on timing used for other antibody therapeutics.

Combinations

In some embodiments, the active agents described herein are administeredalone, and in alternative embodiments, the active agents describedherein are administered in combination with another therapeutic agent,e.g., another active agent of the present disclosures of different type(e.g., structure), or another therapeutic which does not inhibit abinding interaction between EGFR and HSP90. In some aspects, the othertherapeutic aims to treat or prevent cancer. In specific aspects, theother therapeutic is one listed under the section entitled “HeterologousMoieties: Therapeutic Agents.” In some embodiments, the othertherapeutic is a chemotherapeutic agent. In some aspects, thechemotherapeutic agent is a DNA crosslinker or an agent that targets DNAsynthesis (e.g., cisplatin). In some aspects, the chemotherapeutic agentcomprises any of a platinum coordination compound (e.g., cisplatin),topoisomerase inhibitor (e.g., camptothecin), antibiotic compound (e.g.,doxorubicin, mitomycin, bleomycin, daunorubicin, streptozocin), anantimitotic alkaloid (e.g., vinblastine, vincristine, videsine, Taxol,vinorelbine), or an anti-viral (e.g., gemcitabine). In some embodiments,the other therapeutic is an agent used in radiation therapy for thetreatment of cancer. In exemplary aspects, the radiation therapycomprises photon beams (e.g., X rays, gamma rays), electron beams and/orcharged particle beams. In certain aspects, the radiation therapycomprises external beam radiation therapy (e.g., 3-dimensional conformalradiation therapy (3D-CRT), intensity-modulated radiation therapy(IMRT), image-guided radiation therapy (IGRT), tomotherapy, stereostaticradiosurgery, stereostatic body radiation therapy, proton therapy, andthe like). In alternative aspects, the radiation therapy comprisesinternal radiation therapy (a.k.a., brachytherapy), such as,interstitial brachytherapy. In some aspects, the radiation therapycomprises systemic radiation therapy, e.g., ibritumomab tiuxetan(Zevalin®), tositumomab and iodine I 131 tositumomab (Bexxar®),samarium-153-lexidronam (Quadramet®) and strontium-89 chloride(Metastron®).

In exemplary embodiments, the active agent is administeredsimultaneously as the other therapeutic. In alternative embodiments, theactive agent is administered either before or after the othertherapeutic.

Methods of Inhibiting a Binding Interaction Between EGFR and HSP90

Given the importance of the biological roles of EGFR and HSP90,individually, and as shown herein for the first time, in combinationwith one another, the active agents of the present disclosures areuseful for a number of applications in a variety of settings. Forexample and most simplistically, the active agents of the presentdisclosures are useful for inhibiting a binding interaction between EGFRand HSP90 in a cell. In this regard, the present disclosures provide amethod of inhibiting a binding interaction between EGFR and HSP90 in acell. The method comprises contacting the cell with a compound of thepresent disclosures, a pharmaceutically acceptable salt thereof, aconjugate comprising the compound, or a multimer or dimer comprising thecompound, in an amount effective to inhibit the binding interaction. Insome aspects, the cell is part of an in vitro or ex vivo cell culture orin vitro or ex vivo tissue sample. In some aspects, the cell is an invivo cell. In certain embodiments, the method is intended for researchpurposes, and, in other embodiments, the method is intended fortherapeutic purposes.

Methods of Increasing EGFR Degradation

As shown herein for the first time, inhibition of the bindinginteraction between EGFR and HSP90 leads to an increase in EGFRdegradation. Accordingly, the present disclosures further provides amethod of increasing EGFR degradation in a cell. The method comprisescontacting the cell with a compound of the present disclosures, apharmaceutically acceptable salt thereof, a conjugate comprising thecompound, or a multimer or dimer comprising the compound, in an amounteffective to increase the degradation. In some aspects, the cell is partof an in vitro or ex vivo cell culture or in vitro or ex vivo tissuesample. In some aspects, the cell is an in vivo cell. In certainembodiments, the method is intended for research purposes, and, in otherembodiments, the method is intended for therapeutic purposes.

Methods of Treating Cancer

As shown herein for the first time, a compound that inhibits a bindinginteraction between EGFR and HSP90 increases tumor cell death. Thus, thepresent disclosures provides a method of increasing tumor cell death ina subject. The method comprises administering to the subject a compoundof the present disclosures, a pharmaceutically acceptable salt thereof,a conjugate comprising the compound, or a multimer or dimer comprisingthe compound, in an amount effective to increase tumor cell death.

In accordance with the foregoing, the present disclosures furtherprovides a method of treating a cancer in a subject. The methodcomprises administering to the subject a compound of the presentdisclosures, a pharmaceutically acceptable salt thereof, a conjugatecomprising the compound, or a multimer or dimer comprising the compound,in an amount effective to treat the cancer in the subject.

As used herein, the term “treat,” as well as words related thereto, donot necessarily imply 100% or complete treatment. Rather, there arevarying degrees of treatment of which one of ordinary skill in the artrecognizes as having a potential benefit or therapeutic effect. In thisrespect, the methods of treating cancer of the present disclosures canprovide any amount or any level of treatment of cancer. Furthermore, thetreatment provided by the method of the present disclosures may includetreatment of one or more conditions or symptoms of the cancer, beingtreated. Also, the treatment provided by the methods of the presentdisclosures may encompass slowing the progression of the cancer. Forexample, the methods can treat cancer by virtue of reducing tumor orcancer growth, reducing angiogenesis, reducing metastasis of tumorcells, increasing cell death of tumor or cancer cells, and the like.

Methods of Sensitizing Tumors

The present disclosures furthermore provides a method of sensitizing atumor to chemotherapy, radiation therapy, or both chemotherapy andradiation therapy, in a subject. The method comprises administering tothe subject a compound of the present disclosures, a pharmaceuticallyacceptable salt thereof, a conjugate comprising the compound, or amultimer or dimer comprising the compound, in an amount effective tosensitize the tumor to the therapy. As used herein, the term “sensitize”refers to rendering the tumor more treatable by the therapy, such thatthe therapy achieves a greater therapeutic index or efficacy. In someaspects, the chemotherapy comprises any of the chemotherapeuticsdescribed herein, including, but not limited to, a platinum coordinationcompound (e.g., cisplatin), topoisomerase inhibitor (e.g.,camptothecin), antibiotic compound (e.g., doxorubicin, mitomycin,bleomycin, daunorubicin, streptozocin), an antimitotic alkaloid (e.g.,vinblastine, vincristine, videsine, Taxol, vinorelbine), or ananti-viral (e.g., gemcitabine). In some aspects, the radiation therapyis any of those described herein.

In some embodiments, the compound of the present disclosures, apharmaceutically acceptable salt thereof, a conjugate comprising thecompound, or a multimer or dimer comprising the compound, isadministered to the subject simultaneously with the chemotherapy and/orradiation therapy. In some embodiments, the compound of the presentdisclosures, a pharmaceutically acceptable salt thereof, a conjugatecomprising the compound, or a multimer or dimer comprising the compound,is administered to the subject before the chemotherapy and/or radiationtherapy. In particular aspects, the time of administration of thecompound of the present disclosures, a pharmaceutically acceptable saltthereof, a conjugate comprising the compound, or a multimer or dimercomprising the compound, and the time of administration of thechemotherapy and/or radiation therapy are about 1 week or less apart,e.g., about 6 days or less apart, about 5 days or less apart, about 4days or less apart, about 3 days or less apart, about 48 hours or lessapart, about 24 hours or less apart, about 12 hours or less apart, about8 hours or less apart, about 6 hours or less apart, about 4 hours orless apart, about 3 hours or less apart, about 2 hours or less apart,about 1 hour or less apart, about 45 minutes or less apart, about 30minutes or less apart, about 15 minutes or less apart.

Cancer

The cancer treatable by the methods disclosed herein may be any cancer,e.g., any malignant growth or tumor caused by abnormal and uncontrolledcell division that may spread to other parts of the body through thelymphatic system or the blood stream. In some embodiments, the cancer isa cancer in which an EGFR and an HSP90 are expressed by the cells of thecancer. In some aspects, the cancer is a cancer in which an EGFR proteinis over-expressed, the gene encoding EGFR is amplified, and/or an EGFRmutant protein (e.g., truncated EGFR, point-mutated EGFR) is expressed.In some aspects, the cancer is a cancer in which a k-Ras protein isover-expressed, a gene encoding the k-Ras protein is amplified, and/or ak-Ras mutant protein (truncated k-Ras, point-mutated k-Ras) isexpressed.

The cancer in some aspects is one selected from the group consisting ofacute lymphocytic cancer, acute myeloid leukemia, alveolarrhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer ofthe anus, anal canal, or anorectum, cancer of the eye, cancer of theintrahepatic bile duct, cancer of the joints, cancer of the neck,gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear,cancer of the oral cavity, cancer of the vulva, chronic lymphocyticleukemia, chronic myeloid cancer, colon cancer, esophageal cancer,cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma,hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lungcancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynxcancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer,peritoneum, omentum, and mesentery cancer, pharynx cancer, prostatecancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)),small intestine cancer, soft tissue cancer, stomach cancer, testicularcancer, thyroid cancer, ureter cancer, and urinary bladder cancer. Inparticular aspects, the cancer is selected from the group consisting of:head and neck, ovarian, cervical, bladder and oesophageal cancers,pancreatic, gastrointestinal cancer, gastric, breast, endometrial andcolorectal cancers, hepatocellular carcinoma, glioblastoma, bladder,lung cancer, e.g., non-small cell lung cancer (NSCLC),bronchioloalveolar carcinoma.

Subjects

In some embodiments of the present disclosures, the subject is a mammal,including, but not limited to, mammals of the order Rodentia, such asmice and hamsters, and mammals of the order Logomorpha, such as rabbits,mammals from the order Carnivora, including Felines (cats) and Canines(dogs), mammals from the order Artiodactyla, including Bovines (cows)and Swines (pigs) or of the order Perssodactyla, including Equines(horses). In some aspects, the mammals are of the order Primates,Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans andapes). In some aspects, the mammal is a human. In some aspects, thehuman is an adult aged 18 years or older. In some aspects, the human isa child aged 17 years or less.

Kits

In some embodiments, the composition comprising a compound of thepresent disclosures, a pharmaceutically acceptable salt thereof, aconjugate comprising the compound, or a multimer or dimer comprising thecompound, is provided as a kit or package or unit dose. “Unit dose” is adiscrete amount of a therapeutic composition dispersed in a suitablecarrier. Accordingly, provided herein are kits comprising a compound ofthe present disclosures, a pharmaceutically acceptable salt thereof, aconjugate comprising the compound, or a multimer or dimer comprising thecompound.

In some embodiments, the components of the kit/unit dose are packagedwith instructions for administration to a patient. In some embodiments,the kit comprises one or more devices for administration to a patient,e.g., a needle and syringe, a dropper, a measuring spoon or cup or likedevice, an inhaler, and the like. In some aspects, the compound of thepresent disclosures, a pharmaceutically acceptable salt thereof, aconjugate comprising the compound, or a multimer or dimer comprising thecompound, is pre-packaged in a ready to use form, e.g., a syringe, anintravenous bag, an inhaler, a tablet, capsule, etc. In some aspects,the kit further comprises other therapeutic or diagnostic agents orpharmaceutically acceptable carriers (e.g., solvents, buffers, diluents,etc.), including any of those described herein. In particular aspects,the kit comprises a compound of the present disclosures, apharmaceutically acceptable salt thereof, a conjugate comprising thecompound, or a multimer or dimer comprising the compound, along with anagent, e.g., a therapeutic agent, used in chemotherapy or radiationtherapy.

The following examples are given merely to illustrate the presentinvention and not in any way to limit its scope.

EXAMPLES Example 1

The following materials and methods were used in the studies describedin Example 2.

Materials

Geldanamycin (GA) was acquired from Assay Designs. EGFR (sc-03) antibodywas acquired from Santa Cruz Biotechnology. Antibodies forglyceraldehyde-3-phosphate dehydrogenase (GAPDH), HSP70, cleaved PARP,and LC3B were purchased from Cell Signaling Technology, whereas,antibodies to detect ErbB2 and HSP90 were purchased from Neomarkers andPharmingen, respectively. Cycloheximide and MG132 were obtained fromSigma. Peptides were synthesized by Peptide 2.0 and American PeptideCompany. Peptide transfection reagent chariot was purchased from ActiveMotif.

Cell Culture

All the cells were purchased from the American Type Culture Collectionunless otherwise mentioned. The human head and neck squamous cellcarcinoma cell lines UMSCC1, 10B, 11B, 12, 17B, 29, 33 and 74B werekindly provided by T. Carey (University of Michigan, Ann Arbor, Mich.).The lung cancer cell line H1975 was provided by J. Engelman(Massachusetts General Hospital, MA). All cell lines were grown in RPMI1640 supplemented with 10% cosmic calf serum. For all in vitroexperiments, cells were released from flasks using PBS containing 0.01%trypsin and 0.20 mmol/L EDTA, and 6×10⁵ cells were plated onto 100-mmculture dishes two days before any treatment.

Immunoblotting

Cells were scraped into PBS containing a sodium orthovanadate andprotease inhibitor mixture (Roche Diagnostic Co). Cells were incubatedfor 15 min on ice in Laemmli buffer (63 mM Tris-HCl, 2% (w/v) SDS, 10%(v/v) glycerol, and 0.005% (w/v) bromophenol blue) containing 100 mMNaF, 1 mM Na₃Vo₄, 1 mM phenylmethylsulfonyl fluoride, and 1 μg/mlaprotinin. After sonication, particulate material was removed bycentrifugation at 13,000 rpm for 15 min at 4° C. The soluble proteinfraction was heated to 95° C. for 5 min, then applied to a 4-12%bis-tris precast gel (Invitrogen) and transferred onto a PVDF membrane.Membranes were incubated for 1 h at room temperature in blocking bufferconsisting of 3% BSA and 1% normal goat serum in Tris-buffered saline[137 mM NaCl, 20 mM Tris-HCl (pH 7.6), 0.1% (v/v) Tween 20]. Membraneswere subsequently incubated overnight at 4° C. with 1 μg/ml primaryantibody in blocking buffer, washed, and incubated for 1 h withhorseradish peroxidase-conjugated secondary antibody (Cell Signaling,Danvers, Mass.). After three additional washes in Tris-buffered saline,bound antibody was detected by enhanced chemiluminescence plus reagent(Amersham Biosciences, Piscataway, N.J.). For quantification of relativeprotein levels, immunoblot films were scanned and analyzed using ImageJ1.32j software (NIH, Bethesda, Md.). Unless otherwise indicated, therelative protein levels shown represent a comparison to untreatedcontrols.

Immunoprecipitation

Cells were trypsinized, washed twice with 1×PBS, and cell lysates wereprepared by incubation for 30 min on ice in fresh lysis buffer [1%Triton X-100, 0.1% sodium dodacyl sulfate, 0.15M sodium chloride, 0.01Msodium phosphate, pH 7.2 1 mmol/L phenylmethylsulfonyl fluoride, 2 μg/mLaprotinin, 0.2 mmol/L sodium orthovanadate, 50 mM sodium fluoride, 2 mMEDTA] containing 20 mM ammonium molybdate. Immunoprecipitation of EGFRand HSP90 was performed as described previously.³⁴

Site Directed Mutagenesis of EGFR Constructs and Transfections

A modified site directed mutagenesis protocol was used to create thedesired mutations in EGFR. The protocol includes 5′ end phosphorylationof the primer using T4 polynucleotide kinase enzyme followed bypolymerase chain reaction (PCR) with single primer and DpnI enzymetreatment. Primers for site directed mutagenesis were designed byintroducing minimal nucleotide changes in the DNA sequence of EGFRcloned into the N1-EYFP vector (Clontech). Mutations in EGFR wereconfirmed by the University of Michigan DNA sequencing core facility.UMSCC11B and CHO cells were transiently transfected with the constructsusing Lipofectamine (Invitrogen) according to the instructions of themanufacturer.

Clonogenic Cell Survival Assay

Clonogenic assays were performed using standard techniques.³⁴ Thefraction surviving each treatment was normalized to the survival of thecontrol cells. Peptide cell survival curves were fitted using theequation SF=(C₅₀)^(m)/[(C₅₀)^(m)+C^(m)], where SF is the survivingfraction, C is the peptide concentration, C₅₀ is the concentration ofpeptide that produces a 50% cell survival and m is the slope of thesigmoid curve.

Immunostaining

After slides were deparaffinized in xylene and rehydrated using serialethanol dilutions, antigen site unmasking was performed by immersingslides in 100 nmol/L citrate buffer for 20 minutes at high pressure andtemperature inside a pressure cooker. Slides were then washed in PBS,blocked for 1 hour, and incubated in primary antibody at 4° C.overnight. Slides were then washed again in PBS, incubated in secondaryantibody for 1 hour, rewashed, and prepared with a coverslip after adrop of ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole(Molecular Probes) was added to each sample. Fluorescence images wereacquired using a DP70 camera fitted on an Olympus 1X-71 microscope.

Half Life Studies of WT-EGFR and 768-773 EGFR Constructs

CHO cells were transfected with an equal amount of DNA template (1 μg)of WT and 768-773 EGFR constructs. 24 hours post transfection, CHX (50μg/ml) was added to cells expressing each of these constructs. Cellswere harvested at 0, 2 and 6 hours post treatment, and immunoblottingwas carried out for EGFR and GAPDH to analyze the relative decrease inEGFR protein levels in the 768-773 construct compared with WT.

GST EGFR-HSP90 Direct Interaction Assay

GST pull-down experiments to detect a physical interaction between EGFRand HSP90 were performed using standard procedures. Briefly, purifiedGST-EGFR(His672-Ala1210, 90 kDa, 1 μg) fusion protein (Cat #7706, CellSignaling) was incubated with 50 μl (3.5 mg swelled in deionized water)of glutathione-agarose beads (Cat #G4510, Sigma) equilibrated in 0.5×Superdex buffer (1× Superdex buffer: 25 mM HEPES, pH 7.5, 12.5 mM MgCl₂,10 μM ZnSO₄, 150 mM KCl, 20% glycerol, 0.1% Nonidet P-40, and 1 mM EDTA)for 2 hours at 4° C. and then washed three times with 0.5× Superdexbuffer. About 200 ng purified HSP90 protein (Cat # SPP-770, AssayDesigns) was then added to the washed beads and incubated overnight at4° C. The beads were washed three times using 0.5 Superdex buffer,boiled in Laemmli buffer, and the bound HSP90-EGFR complex wasimmunodetected following immunoblotting with HSP90 and EGFR specificantibodies.

Peptide Internalization Assay

The UMSCC1 xenografts were cryopreserved in OCT (Electron MicroscopySciences). Five μm sections were cut from tissues prepared on days 1 and3 after peptide treatment. Sections were fixed in cold methanol for 20min and then were blocked with 1% Bovine Serum Albumin (BSA). Thesections were incubated overnight with anti-EGFR antibody (Santa Cruz).After washing, the sections were incubated with anti-rabbit Alexa Fluor488 (Molecular Probes) and Streptavidin conjugated Alexa Fluor 594(Molecular Probes). Coverslips were mounted with one drop of ProlongGold anti-fade reagent with DAPI (4,6-diamidino-2-phenylindole;Molecular Probes) to visualize the nuclei. Fluorescence images wereacquired using a DP70 camera fitted on an Olympus 1X-71 microscope.

Peptide Binding Assay

UMSCC1 cells (1×10⁶) were scraped in PBS containing protease andphosphatase inhibitors, then frozen and thawed three times using dryice. Next, supernatant was collected and incubated with 3 or 10 μg/ml ofbiotinylated peptide for 1 hour at 37° C., followed by the addition of20 μl of Streptavidin agarose beads (EZview red Streptavidin affinitygel, Sigma). Samples were rotated overnight at 4° C., boiled in 30 μl ofLaemmli buffer at 100° C. for 10 minutes, and immunoblotted for HSP90 todetect binding with peptide.

ATP Binding Assay

Cell lysates were prepared in RIPA buffer. About 500 μg protein wasincubated overnight at 4° C. with 25 μl of γ-linked ATP-Agarose beads(Innova Biosciences). After centrifugation, beads were washed in PBS 6times and ATP bound proteins were extracted in Laemilli buffer, resolvedon a SDS page and immunoblotted with anti-HSP90 antibody to detectchange in ATP bound HSP90 levels.

In Vivo Tumor Growth Studies

Mice were handled according to the established procedures of theUniversity of Michigan Laboratory Animals Maintenance Manual. Togenerate tumor xenografts, 2×10⁶ UMSCC1 cells were transplanted into theflanks of athymic nude Foxn1^(nu) mice (Harlan Laboratories). Whentumors reached a volume of ˜50 mm³, the mice were randomized into 3groups (1 untreated control and 2 experimental groups for specific andnon-specific peptides) containing from 15 to 25 tumors, and treatmentwas initiated.

Live Cell Imaging to Monitor Autophagy:

Change in LC3 localization upon EGFR degradation after treatment withpeptide was monitored in live HeLa cells that were stably expressingLC3B-GFP construct. LC3-I is converted to LC3-II during autophagy, andcauses the autophagosomes to appear as punctate spots using afluorescent microscope. HeLa-LC3-GFP cells were plated in opticallyclear multi-chamber slides. After overnight incubation with cellpermeable peptide, change in LC3 expression was monitored. Chloroquine(8 μM) which stabilizes LC3 autophagosomes, was used as a positivecontrol.

Statistics

Tumor volume doubling was determined for each xenograft by identifyingthe earliest day on which it was at least twice as large as on the firstday of treatment. A cubic smoothing spline was used to obtain the exacttime of doubling, and the Kaplan-Meier method was used to analyze thedoubling times derived from the smoothed growth curves. The log ranktest was used for comparisons between any two treatment groups. Resultsare presented as mean±SEM, and Student's t test was used to assess thestatistical significance of differences. A significance level thresholdof P<0.05 was used.

Example 2

The following studies were carried out using the materials and methodsdescribed Example 1.

EGFR Physically Interacts with HSP90

To determine whether EGFR directly interacts with HSP90, 11 cancer and 2normal cell lines that express different levels of EGFR were selectedand immunoprecipitation of HSP90 followed by immunoblotting for EGFR wasperformed. It was found that EGFR was consistently immunoprecipitatedwith HSP90 (FIG. 1 a, and FIG. 2) although the degree of interactionvaried across these lines. This interaction was confirmed byimmunoprecipitating EGFR and immunoblotting with HSP90 antibody (FIG.2). To further confirm EGFR-HSP90 interaction, a FLAG-tagged HSP90 wasexpressed in UMSCC11B cells, immunoprecipitated FLAG and immunoblottedfor EGFR. It was found that ectopically expressed HSP90 also interactedwith endogenous EGFR (FIG. 1 b). This interaction was reduced upontreatment with geldanamycin (GA), an inhibitor of HSP90 activity. Sinceboth EGFR and HSP90 are known to interact with ErbB2, we next sought todetermine whether the EGFR-HSP90 interaction was direct or via ErbB2.When we expressed full length EGFR in CHO cells, which are ErbB2negative, EGFR was immunoprecipitated using HSP90 antibody (FIG. 1 c).Likewise, GST pull-down assays demonstrated a direct physicalinteraction between EGFR and HSP90 (FIG. 1 d). These results show thatthe EGFR-HSP90 interaction is direct and not mediated byheterodimerization of EGFR with ErbB2.

Identification of a Potential Binding Region of HSP90 on EGFR

Because the interaction between ErbB2 and HSP90 involves the kinasedomain of ErbB2¹⁶, we hypothesized that the kinase domain of EGFR mightalso contain the HSP90 binding region. Therefore, we constructed mutantsof EGFR based on the binding region of ErbB2 with HSP90¹⁶, expressedthem in CHO cells and assessed their interactions with HSP90. We beganwith a construct with six substitutions in this region (768-773 EGFR;SVDNPH (SEQ ID NO: 15) to NHVPSD (SEQ ID NO: 35)) by scrambling theamino acids from the native sequence. There was almost no interactionbetween HSP90 and the 768-773 EGFR construct (FIG. 3 a). We thenconstructed mutants with only single and double substitutions (S768A,S768D, S768I, D770G, DN770-771AA, and P772G). Immunoprecipitation ofHSP90 revealed that S768D and S768I, D770G and P772G -EGFR had a similarinteraction with HSP90 as WT-EGFR. We found a decrease in interactionwith HSP90 in the S768A-EGFR expressing cells. The interaction wasslightly decreased in the case of the double mutant DN770-771AA (FIG. 3b). These results demonstrate that the interaction between EGFR andHSP90 does not depend on a single amino acid but on the 6 amino acidsfrom 768 to 773.

As we expected HSP90 to stabilize EGFR, we anticipated that EGFRexpression would correlate with HSP90 binding. Indeed, the twoconstructs S768A and DN770-771AA were expressed in lower amountscompared to the WT-EGFR, whereas the 768-773 EGFR construct that showsminimum interaction with HSP90 was expressed at significantly lowerlevels than either single or double EGFR mutants. However, we needed torule out the possibility that this finding was due to the decreasedexpression of EGFR. We hypothesized that if decreased EGFR levels incells transfected with the 768-773 EGFR construct were due to a decreasein its interaction with HSP90, then restoration of EGFR levels byinhibiting degradation would not restore the EGFR-HSP90 interaction. Weexpressed the 768-773 EGFR and WT-EGFR construct in CHO cells, treatedcells with a proteasomal inhibitor MG132 (5 μM)) for 8 hours, andassessed whether restoration of EGFR levels via blockade of proteindegradation caused any change in the EGFR-HSP90 interaction. We foundthat MG132 pretreatment caused a significant increase in 768-773 EGFRlevels (FIG. 3 c), but that the interaction of expressed 768-773 EGFRwith HSP90 still remained low when compared to the WT-EGFR. To furthertest our hypothesis that 768-773 EGFR might be vulnerable to degradationdue to its decreased binding with HSP90, we expressed the WT and 768-773EGFR constructs in CHO cells and treated with cycloheximide (CHX, 50μg/ml) to block protein synthesis. EGFR levels were reduced by 30% inWT-EGFR expressing cells at 3 hours and by 50% at 6 hours. However,levels of 768-773 EGFR were reduced by about 90% within 3 hours, showingthat 768-773 EGFR is a significantly less stable protein than WT-EGFR(FIG. 3 d). These results demonstrate that the decrease in 768-773EGFR-HSP90 interaction is not due simply to lowered expression of768-773 EGFR.

Specificity and Efficacy of EGFR-HSP90 Interaction Inhibitory Peptide

We next sought to target EGFR for degradation by use of peptides thatwould competitively inhibit EGFR-HSP90 binding. We also focused on alarger region of 21 amino acids, considering residues flanking the768-773 site (Table 1).

TABLE I Effect of synthetic peptides on clonogenic survival of UMSCC1cells: Peptide Sequence Survival Fraction 1 DEAYVMA 0.93 ± 0.1  2SVDNPHVC 0.77 ± 0.12 3 RLLGIC 0.85 ± 0.16 4 GVGSPYVS    1 ± 0.16 5DEAYVMASVDNPHVCRLLGIC    1 ± 0.14 6 DEAYVMAGVGSPYVSRLLGIC    1 ± 0.12 7SVGNPHVC 0.97 ± 0.21 8 NHVPSDVC   1 ± 0.1

We treated UMSCC1 cells with these peptides using Chariot (a peptidedelivery agent from Active Motif, Carlsbad, Calif.) and assessed EGFRdegradation (data not shown). We found that peptides 2 and 3, which aredirected at the 768-773 of EGFR, caused a decrease in cell survival(Table 1), confirming the importance of this domain for EGFR-HSP90interaction.

We selected peptide #2 (named “Disruptin”) and the scrambled peptide(peptide #8) as a control for further studies. These two peptides weresynthesized along with 11 amino acids selected from the HIV-TAT gene toenable cellular uptake, and a biotin moiety was attached for molecularstudies^(17,18) (Table 1b).

TABLE II Design and sequence of cell permeable peptides and theireffects on clonogenic survival in different cancer and normal celllines. a Specific Peptide Biotin-YGRKKRRORRR-SVDNPHVC Non SpecificPeptide Biotin-YGRKKRRORRR-NHVPSDVC b Human Cancer H&N Lung UMSCC1UMSCC10B UMSCC74B A549 H1975 WT-EGFR WT-EGFR WT-EGFR WT-EGFR T790M-EGFRSpecific 0.76 ± 0.02 0.63 ± 0.06 0.74 ± 0.15 0.57 ± 0.11 0.40 ± 0.08 NonSpecific 1.62 ± 0.04 0.98 ± 0.04 0.88 ± 0.10 0.81 ± 0.09 0.77 ± 0.17 bHuman Hamster Cancer Normal Lung Cervical Epithelial Fibroblasts OvarianH3255 HeLa Het1A MRC5 CHO L858R-EGFR WT-EGFR WT-EGFR WT-EGFR EGFRSpecific 0.71 ± 0.05 0.72 ± 0.05 1.02 ± 0.03 1.01 ± 0.04 0.98 ± 0.02 NonSpecific  1.0 ± 0.07 0.92 ± 0.08 0.91 ± 0.11 1.04 ± 0.05 1.12 ± 0.14

Uptake and binding studies revealed that the peptides were stable for upto 24 hours (FIG. 4). We then assessed colonogenic survival in head andneck (UMSCC1, UMSCC10B, UMSCC74B), lung (A549, H1975, H3255), and HeLacancer cells. Normal human esophageal squamous epithelial (Het1A) andlung fibroblast (MRC5) cell lines along with EGFR negative CHO cellswere selected to study selectivity. The EGFR specific peptide reducedthe surviving fraction of the cancer cell lines containing WT-EGFR,including UMSCC1 (0.76±0.02), UMSCC10B (0.63±0.06), UMSCC74B (0.74±0.15)HeLa (0.72±0.05), and A549 (0.57±0.11), as well as in H1975, whichcontains the T790M mutation and is resistant to erlotinib, (0.40±0.08)and H3255 (containing the L858R EGFR mutation and is sensitive toerlotinib, 0.71±0.05) (Table 2b). The peptide did not affect the normalcell lines.

The specific peptide directly interacts with HSP90 and disrupts theEGFR-HSP90 interaction

To determine whether the EGFR specific peptide directly interacts withHSP90, we carried out affinity purification of the biotinylated-peptidesin two cancer (UMSCC1, H1975) and two normal cell lines (Het1A andMRC5). HSP90 protein binding was increased (5 to 10 fold) only with theEGFR specific peptide in cancer cells but not in the normal cells (FIG.5 a). We also found a concentration-dependent increase in the binding ofthe specific peptide with HSP90 (FIG. 6 a). Next, we sought to determinewhether the specific peptide disrupted EGFR-HSP90 interactions in tumorcells relative to normal cells. The HSP90 inhibitor geldanamycin (GA)(50 nM), was used as positive control. After a 24-hour exposure tospecific peptide, EGFR-HSP90 interaction was significantly reduced inthe cancer cell lines, which was comparable with GA treatment, but therewas no effect in response to the non-specific peptide in the tumor cellsor specific peptide in normal cells (FIG. 5 b). Peptide treatmentaffected neither HSP90 activity as assessed by HSP90 and ATP binding inUMSCC1 cells (FIG. 6 b), nor the levels of either HSP90 or HSP70, bothof which were elevated by the HSP90 ATPase inhibitor GA (FIG. 5 b). Wesubsequently observed a loss of EGFR protein (FIG. 5 c) and clonogenicsurvival (FIG. 5 d and Table II) in tumor cells at 72 h post treatment.These findings demonstrate that the specific peptide binds with HSP90,disrupts EGFR-HSP90 interaction, decreases EGFR stability of both wildtype and erlotinib resistant T790M-EGFR, and decreases clonogenicsurvival, recapitulating the effect of 768-773 mutation on EGFR.

Effects of the Specific Peptide on EGFR Levels in Human Xenografts

To determine the effects of the specific peptide on EGFR levels in vivo,we injected these peptides into nude mice bearing established UMSCC1xenografts. We found that specific peptides were detectable inxenografts as early as one day and remained detectable 72 hours postinjection (FIG. 7) and caused a reduction in EGFR immunostaining. Todetermine the specificity and long term effects of the EGFR specificpeptide, we harvested tumors along with normal adjacent tissue on day 18post treatment. EGFR, but not HSP90, staining was decreased in sectionsfrom the tumor xenografts of specific-peptide treated mice, but wasunchanged in the adjacent normal tissue (FIG. 8 a). These resultssuggest that although the EGFR kinase domain is 100% conserved betweenmouse and human, the peptide preferentially targets EGFR in the tumorcells and does not seem to affect EGFR in the normal cells.

Tumor lysates were also analyzed for the levels of EGFR, HER2, HER3,pERK, tERK, HSP90 and GAPDH. Disruptin treatment caused a markedreduction in EGFR and pERK levels, whereas, HER2, HER3 levels werelargely unaffected.

We then assessed the effect of the peptides on tumor growth in UMSCC1xenografts (FIGS. 8 b and c). A single dose of specific peptidesignificantly increased median tumor doubling time (16 days) comparedwith both mock treated and non-specific peptide treated mice (5.5 days)(P=0.0002) (data not shown). The administration of two injections of thepeptide (3 days apart) significantly increased median tumor doublingtime (>22 days) compared with the mock-treated group (5.5 days)(P<0.0001) (FIG. 8 d). No systemic toxicity was observed. We found thatthere was no difference between any of the treatment groups in eitherKi67 or ApopTag staining 18 days after treatment (FIG. 9 a), suggestingthat the tumor response was independent of cell proliferation orapoptosis. We then stained these samples with LC3B antibody, which is amarker for autophagic death. We found an increase in LC3B punctatestaining in tumors that were treated with specific peptide compared toeither non-specific peptide or DMSO treated control. These findings werefurther confirmed in cultured HeLa cells that stably express LC3B-GFPfusion protein (FIG. 9 b), suggesting that the response is due to aninduction of autophagy. The effect of Disruptin treatment on micro-bloodvessel density in the samples was analyzed by CD31 immunostaining and isshown in FIG. 10.

The effect of Disruptin on tumor growth in NCI-H1975 xenografts was alsoassessed in comparison to erlotinib. To generate tumor xenografts, 2×10⁶NCI-H1975 cells were transplanted into the flanks of athymic nudeFoxn1^(nu) mice (Harlan Laboratories). When tumors reached a volume ofabout 50 mm³, the mice were randomed into treatment groups and Disruptintreatment as described for the UMSCC1 xenograft mice above was initiatedexcept that the NCI-H1975 bearing animals were also treated daily witherlotinib (100 mg/kg, p.o., one week). As shown in FIG. 11 a-c,Disruptin also increased tumor doubling time in the NCI-H1975 bearinganimals.

Example 3

To assess the efficacy of a peptide of the present disclosures againsttumors expressing either wild-type or erlotinib-resistant EGFR, grown asxenografts, four different cell lines that contain either WT or mutantEGFR are implanted into the mice of a treatment group (10 mice pergroup) to produce xenografts. A total of 160 mice are used (4 celllines×4 treatment groups×10 mice per group). Each mouse is prepared withtwo tumors. Once the tumor has reached about 100 mm³, mice arerandomized into 4 groups containing at least 10 animals per group. Miceare then given (a) specific peptide (8 mg/kg, ip, day 1 and 3) (b)non-specific peptide (same as specific peptide), (c) erlotinib (80mg/kg, oral, 5 doses), (d) DMSO (same as specific peptide). Three miceare sacrificed to take out 6 tumors on day 3 and 6 tumors on day 18 toassess the effect on EGFR-HSP90 interaction by immunoprecipitation andon EGFR degradation by immunoblotting and immunofluorescence analysis ofEGFR and key down-stream signaling molecules, such as pAKT, pERK1/2 (asdescribed previously (Nyati et al., Clin Cancer Res 10: 691-700 (2004)).The effect of treatment on apoptosis and cell proliferation are assessedby TUNEL and Ki-67 staining. The remaining tumors are monitored forgrowth for 60 days or until tumors have reached a maximum of 1 cm×1 cm.

The median growth rate or time to doubling for each cell line in eachtreatment is determined. Linear contrasts are used to test the effect oftreatment on growth rates overall, and within cell line types. Coxproportional hazards regression models are utilized to test fordifferences in doubling times while allowing inclusion of data fromtumors removed on days 3 and 18 (censored if volume not doubled at timeof removal). Treatment, cell line and their interaction are included ascovariates. An overall comparison between two treatment groups isobtained by stratifying the analysis on cell line. Pairwise comparisonsof particular interest include peptide vs. control, peptide+cisplatinvs. cisplatin and peptide+cisplatin vs. peptide.

Example 4

To assess the efficacy and mechanism of EGFR degradation induced bytreatment with a compound of the present disclosures, and the role ofthis treatment in chemo and radiosensitivity, two cell lines fromExample 3 are selected: one that expresses WT-EGFR and second thatexpresses T790M-EGFR for combination studies. Mice are prepared asdescribed in Example 3 and are given a combination treatment whereineither a control peptide or a peptide compound of the presentdisclosures are administered followed by a single dose of cisplatin (5mg/kg), or 5 radiation treatments (i.e. Mon-Fri, 2Gy per fraction). Inthis experiment, each group will contain at least 10 mice per group and180 mice in total are used (2 cell lines×9 treatment groups×10 mice pergroup). Three mice are sacrificed on days 3 and 18 to assess the effecton EGFR-HSP90 interaction by immunoblotting, immunoprecipitation and byimmunofluorescence analysis as described in Example 3. The remainingtumors are monitored for growth for 60 days or until tumors have reacheda maximum of 1 cm×1 cm.

The median growth rate or time to doubling for each cell line in eachtreatment is determined. Linear contrasts are used to test the effect oftreatment on growth rates overall, and within cell line types. Coxproportional hazards regression models are utilized to test fordifferences in doubling times while allowing inclusion of data fromtumors removed on days 3 and 18 (censored if volume not doubled at timeof removal). Treatment, cell line and their interaction are included ascovariates. An overall comparison between two treatment groups isobtained by stratifying the analysis on cell line. Pairwise comparisonsof particular interest include peptide vs. control, peptide+cisplatinvs. cisplatin and peptide+cisplatin vs. peptide.

Example 5

The safety of Disruptin compared to geldanamycin in immune-competentC57BL/6 mice was investigated.

Mice were dosed by intraperitoneal injection with 10 and 30 mg/kg ofDisruptin, and brain, lung, liver, heart, kidneys, spleen, stomach,small intestine, mesenteric lymph nodes, cecum, colon, pancreas,ovaries, bone marrow, and eyes were evaluated 3 days post-injection.There were no histological alterations in the evaluated organs aftertreatment with Disruptin or the scrambled peptide at either dose tested.In contrast, mice treated with geldanamycin showed histological evidenceof toxicity in eye and liver. Complete blood counts and liver cytosolicenzymes (AST, ALT) were not significantly different from controls in theDisruptin and scrambled peptide treated mice at either dose. Theseresults suggest that Disruptin, at an effective dose, was well-toleratedand lacked the adverse effects seen in geldanamycin treated mice.

Example 6

The effect of Disruptin (peptide #2) on capillary sprouting was assessedin human dermal microvascular cells.

The cells were seeded in 24-well plates coated with growth factorreduced Matrigel (BD Biosciences, Bedford, Mass., USA). The wells werethen treated on day 1 with the scrambled peptide (peptide #8) andDisruptin (100 μg/ml). Three days after treatment, the capillarybranches were counted and analyzed. It was found that network haddegenerated in the Disruptin-treated wells. Cell death was assessed byPropidium Iodide staining. FIG. 12 shows the reduced percentage ofcapillary sprouting in Disruptin-treated wells as compared to thescrambled peptide-treated wells.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range and each endpoint, unless otherwise indicatedherein, and each separate value and endpoint is incorporated into thespecification as if it were individually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

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What is claimed is:
 1. A composition comprising a compound that inhibitsa binding interaction between an epidermal growth factor receptor (EGFR)and a heat shock protein 90 (HSP90) wherein the compound consists of theamino acid sequence SVDNPH (SEQ ID NO: 15), or SVDNPHV (SEQ ID NO: 16),or SVDNPHVX (SEQ ID NO: 17) fused to a cell penetrating peptide (CPP).2. A composition comprising a compound that inhibits a bindinginteraction between an epidermal growth factor receptor (EGFR) and aheat shock protein 90 (HSP90) wherein the compound consists of an aminoacid sequence selected from the group consisting of: AVDNPH, (SEQ ID NO:25) DVDNPH, (SEQ ID NO: 26) IVDNPH, (SEQ ID NO: 27) SVGNPH, (SEQ ID NO:28) SVDNGH, (SEQ ID NO: 29) and SVAAPH. (SEQ ID NO: 30)

fused to a cell penetrating peptide (CPP).
 3. A composition comprising acompound that inhibits a binding interaction between an epidermal growthfactor receptor (EGFR) and a heat shock protein 90 (HSP90) wherein thecompound consists of the amino acid sequence XDNPHX (SEQ ID NO: 32)fused to a cell penetrating peptide (CPP).
 4. A composition comprising acompound that inhibits a binding interaction between an epidermal growthfactor receptor (EGFR) and a heat shock protein 90 (HSP90) wherein thecompound consists of the amino acid sequence SXDNPHXX (SEQ ID NO: 33)fused to a cell penetrating peptide (CPP).
 5. The composition of claim1, 2, 3, or 4, comprising an intramolecular bridge which covalentlylinks the side chains of two amino acids of the compound.
 6. Thecomposition of claim 5, wherein the side chains of the amino acids atpositions 1 and 6 are covalently linked by the intramolecular bridge. 7.The composition of claim 1, 2, 3, or 4, comprising a pharmaceuticallyacceptable carrier, diluent, or excipient.
 8. A method of treating asubject comprising administering to the subject a composition of claim 7in an amount effective to treat the subject.
 9. The method of claim 8,wherein the subject is characterized by overexpression of EGFR orexpression of a mutant EGFR.
 10. A method of sensitizing a subject,comprising administering to the subject a composition of claim 1, 2, 3,or 4 in an amount effective to sensitize the subject to a therapy.
 11. Akit comprising the composition of claim 1, 2, 3, or 4 in combinationwith an agent utilized in radiation therapy or chemotherapy.